From the Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, D. C. 20007
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A transient burst of poly(ADP-ribosyl)ation of
nuclear proteins occurs early, prior to commitment to death, in human
osteosarcoma cells undergoing apoptosis, followed by caspase-3-mediated
cleavage of poly(ADP-ribose) polymerase (PARP). The generality of this early burst of poly(ADP-ribosyl)ation has now been investigated with
human HL-60 cells, mouse 3T3-L1, and immortalized fibroblasts derived
from wild-type mice. The effects of eliminating this early transient
modification of nuclear proteins by depletion of PARP protein either by
antisense RNA expression or by gene disruption on various morphological
and biochemical markers of apoptosis were then examined. Marked
caspase-3-like PARP cleavage activity, proteolytic processing of CPP32
to its active form, internucleosomal DNA fragmentation, and nuclear
morphological changes associated with apoptosis were induced in
control 3T3-L1 cells treated for 24 h with anti-Fas and
cycloheximide but not in PARP-depleted 3T3-L1 antisense cells exposed
to these inducers. Similar results were obtained with control and
PARP-depleted human Jurkat T cells. Whereas immortalized PARP +/+
fibroblasts showed the early burst of poly(ADP-ribosyl)ation and a
rapid apoptotic response when exposed to anti-Fas and cycloheximide,
PARP /
fibroblasts exhibited neither the early
poly (ADP-ribosyl)ation nor any of the biochemical or morphological
changes characteristic of apoptosis when similarly treated. Stable
transfection of PARP
/
fibroblasts with wild-type PARP rendered the
cells sensitive to Fas-mediated apoptosis. These results suggest that
PARP and poly(ADP-ribosyl)ation may trigger key steps in the apoptotic
program. Subsequent degradation of PARP by caspase-3-like proteases may
prevent depletion of NAD and ATP or release certain nuclear proteins
from poly(ADP-ribosyl)ation-induced inhibition, both of which might be
required for late stages of apoptosis.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Apoptosis, or programmed cell death, plays important roles in development, homeostasis, and immunological competence. It is characterized by marked morphological changes such as membrane blebbing, chromatin condensation, nuclear breakdown, and the appearance of membrane-associated apoptotic bodies, as well as by internucleosomal DNA fragmentation. The enzyme poly(ADP-ribose) polymerase (PARP)1 catalyzes the poly(ADP-ribosyl)ation of various nuclear proteins with NAD as substrate, and, because it is activated by binding to DNA ends or strand breaks, PARP has been suggested to contribute to cell death by depleting the cell of NAD and ATP (1). PARP undergoes proteolytic cleavage into 89- and 24-kDa fragments that contain the active site and the DNA-binding domain of the enzyme, respectively, during drug-induced apoptosis in a variety of cells (2). More recently, PARP has been implicated in the induction of both p53 expression and apoptosis (3), with the specific proteolysis of the enzyme thought to be a key apoptotic event (4-6).
Caspase-3, a member of the caspase family of 10 or more
aspartate-specific cysteine proteases that play a central role in the
execution of the apoptotic program (7), is primarily responsible for
the cleavage of PARP during cell death (4, 5). Other caspases, such as
caspase-7, also cleave PARP in vivo, but at lower
efficiencies. Composed of two subunits of 17 and 12 kDa that are
derived from a common proenzyme (CPP32), caspase-3 is related to
interleukin-1-converting enzyme and CED-3, which is required for
apoptosis in Caenorhabditis elegans (8). In human osteosarcoma cells that undergo confluence-associated apoptosis over a
10-day period, caspase-3-like activity, measured with a specific
[35S]PARP-cleavage assay in vitro, peaks at
6-7 days after initiation of apoptosis, concomitant with the onset of
internucleosomal DNA fragmentation (4).
We recently examined the time course of PARP activation and cleavage during apoptosis in intact osteosarcoma cells by immunofluorescence microscopy with antibodies to PARP, to the 24-kDa cleavage product of PARP, and to poly(ADP-ribose) (PAR) (9). We observed a transient burst of synthesis of PAR from NAD that increased early and was maximal 3 days after initiation of apoptosis, prior to the appearance of internucleosomal DNA cleavage (at day 7) and before the cells became irreversibly committed to apoptosis. During this early period, expression of full-length PARP was detected by both immunofluorescence and immunoblot analysis. The amounts of both PAR and PARP decreased thereafter, and at 6 days, the 24-kDa cleavage product of PARP was detected both immunocytochemically and by immunoblot analysis. PAR was not observed during days 8 to 10, despite the presence of abundant DNA strand breaks, potential activators of PARP, during this time. These observations suggested that short-lived PARP-catalyzed poly(ADP-ribosyl)ation may be important at an early stage of apoptosis and is followed by the cleavage of PARP by mid-apoptosis. We have now investigated whether this transient poly(ADP-ribosyl)ation occurs in other cell lines and with other inducers of apoptosis. We examined both cell lines stably transfected with inducible PARP antisense constructs (9-11) and immortalized fibroblasts derived from PARP knockout mice (12) to determine the effect of preventing the early burst in PARP activity on specific markers of apoptosis.
Several PARP knockout mice have been established by disrupting the PARP
gene in embryonic stem cells; these animals neither express PARP nor
exhibit any poly(ADP-ribosyl)ation (12, 13). Despite variations in the
physiological phenotypes of these animals, developmental apoptosis
seems to take place in the absence of PARP. Furthermore, primary cells
(fibroblasts, splenocytes, thymocytes) from these animals undergo
apoptosis induced by N-methyl-N-nitrosourea (MNU)
and other agents (13, 14). The more recent PARP knockout mice exhibit
extreme sensitivity to -irradiation and MNU, and primary cells
derived from these mice showed an abnormal apoptotic response to MNU
(13). In contrast, >80% of primary embryonic fibroblasts derived from
the earlier PARP knockout mice lost viability when exposed to anti-Fas
(1000 ng/ml) for 8 h; thus, the absence of PARP does not seem to
interfere with programmed cell death in these primary cells (14).
Our previous studies with clonal cells depleted of PARP by expression of PARP antisense RNA have supported accessory roles for PARP and/or poly(ADP-ribosyl)ation in adipocyte differentiation (11), DNA replication associated with this differentiation (15, 63), genomic stability (10), and DNA repair (33, 16). In DNA repair, for example, although the absence of PARP did not totally prevent the repair of single-strand breaks, it resulted in a significant delay in this process (16). Primary cell cultures presumably consist of a mosaic of different stages of development, many of which perhaps possess compensatory routes to overcome gene disruption. It is possible that biochemical roles, not easily observable in the context of the whole animal or in primary cultures of cells, can be identified in clonal cells because of more profound effects observed in these cells.
The present study demonstrates the occurrence of a transient poly(ADP-ribosyl)ation of nuclear proteins at an early stage of apoptosis induced by serum deprivation, camptothecin, or antibodies to Fas in different cell lines. When this early poly(ADP-ribosyl)ation was prevented as a result of depletion of endogenous PARP, either by gene disruption or by antisense RNA expression, several morphological and biochemical markers of apoptosis were no longer observed in response to such inducers.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture, Vectors, and Transfection--
A 1.1-kilobase
fragment of murine PARP cDNA encoding the DNA-binding domain and
the NH2-terminal automodification domain (for the mouse
3T3-L1 cell transfections) or a 3.7-kilobase XhoI
full-length human PARP cDNA (for human Jurkat T cell transfection)
was subcloned in an antisense orientation in the expression vector
pMAM-neo (CLONTECH) under the control of the
dexamethasone-inducible mouse mammary tumor virus promoter. The
resulting pMAM-As (antisense) or pMAM-neo (control) plasmids were
transfected into cells by calcium phosphate precipitation (3T3-L1
cells) or by electroporation (Jurkat cells). Transfectants were
selected in appropriate medium with G-418 (400 µg/ml) (Life
Technologies, Inc.). Stably transfected 3T3-L1 cells and fibroblasts
derived from both wild-type and PARP knockout mice (12) were cultured
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). The PARP +/+ and /
fibroblasts, immortalized by a
standard 3T3 protocol, were kindly provided by Z. Q. Wang
(International Agency for Research on Cancer, Lyon, France). Jurkat T
cells and HL-60 cells were maintained in RPMI 1640 supplemented with
10% and 20% fetal bovine serum, respectively. PARP
/
fibroblasts
were either cotransfected with a plasmid expressing wild-type PARP
(pcD-12; Ref. 17) along with the plasmid pTracer-CMV (Invitrogen), a
zeocin-based vector system, or with pTracer-CMV alone using
lipofectamine (Life Technologies, Inc.). This vector system was
utilized as the PARP
/
fibroblasts expressed an endogenous neo
gene, which was used to establish the original PARP knockout mice.
Stable transfectants were colony-selected in growth medium containing
500 µg/ml Zeocin.
PARP-cleavage Assay-- In vitro PARP-cleavage assays were performed as described previously (4, 9). In brief, full-length human PARP cDNA was excised from pcD-12 (17), ligated into the XhoI site of pBluescript-II SK+ (Stratagene), and used to synthesize [35S]methionine-labeled PARP by coupled T7 RNA polymerase-mediated transcription and translation in a reticulocyte lysate system (Promega). Cytosolic extracts of various cells were prepared by rapid freezing and thawing of cells in a solution containing 10 mM Hepes-KOH (pH 7.4), 2 mM EDTA, 0.1% CHAPS detergent, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, pepstatin A (10 µg/ml), leupeptin (10 µg/ml), and aprotinin (10 µg/ml), followed by centrifugation of the cell lysate at 100,000 × g for 30 min and recovery of the supernatant. In vitro PARP-cleavage activity was measured in 25-µl reaction mixtures containing 5 µg of cytosolic protein, [35S]PARP (~5 × 104 cpm), 50 mM Pipes-KOH (pH 6.5), 2 mM EDTA, 0.1% CHAPS, and 5 mM dithiothreitol. After incubation for 1 h at 37 °C, reactions were terminated by the addition of SDS sample buffer (4% SDS, 4% 2-mercaptoethanol, 10% glycerol, 125 mM Tris-HCl (pH 6.8), and 0.02% bromphenol blue). Proteins were resolved by SDS-polyacrylamide gel electrophoresis, and PARP-cleavage products were visualized by fluorography.
Indirect Immunofluorescence Microscopy and Immunoblot
Analysis--
The procedures for fixation and staining with monoclonal
antibodies to PAR (10HA) (18) have been described previously (9). Cells
were transferred to a slide in a Cytospin (IEC Centra), fixed with 10%
(w/v) ice-cold trichloroacetic acid for 10 min, and dehydrated in 70%,
90%, and absolute ethanol for 3 min each at 20 °C. The slides
were then incubated overnight in a humid chamber at room temperature
with antibodies to PAR (1:250 dilution) in phosphate-buffered saline
(PBS) containing 12% bovine serum albumin. After washing with PBS,
cells were incubated for 1 h with biotinylated anti-mouse IgG
(1:400 dilution in PBS-bovine serum albumin), washed, and incubated for
30 min with streptavidin-conjugated Texas red (1:800 dilution in
PBS-bovine serum albumin). Cells were finally mounted with PBS
containing 80% glycerol and observed with a Zeiss fluorescence
microscope. All exposure times were identical to allow comparisons of
relative staining intensities at various times during apoptosis.
PARP Activity Assays-- At indicated time intervals, cells were harvested by scraping, washed with ice-cold PBS, and assayed for PARP activity as described previously (20). Briefly, cells were sonicated for 20 s (three times) to lyse cells and introduce DNA strand breaks required for PARP activity, followed by measurement of [32P]NAD incorporation into acid-insoluble acceptors at 25 °C for 1 min.
Detection of Apoptotic Internucleosomal DNA Fragmentation-- Cells were washed in PBS and lysed in 7 M guanidine hydrochloride, and total genomic DNA was extracted and purified using a Wizard Miniprep DNA Purification Resin (Promega). After RNase A treatment (1 µg/50 µl) of the DNA samples for 30 min, apoptotic internucleosomal DNA fragmentation was detected by gel electrophoresis on a 1% agarose gel and ethidium bromide staining (0.5 µg/ml) as described previously (64).
Hoechst Staining for Apoptotic Morphology-- Cells were centrifuged at 1000 rpm for 5 min, fixed for 10 min in PBS containing 4% formalin, washed with PBS, and stained with Hoechst 33258 (24 mg/ml) in PBS containing 80% glycerol. An aliquot (25 µl) of the cell suspension was then dropped onto a slide, and nuclear morphology was observed with an Olympus BH2 fluorescence microscope.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A Transient Burst of Poly(ADP-ribosyl)ation of Nuclear Proteins Occurs during Early Stages of Apoptosis in HL-60 Cells-- To investigate whether the brief activation of PARP during the early stages of apoptosis, detected initially by immunofluorescence in osteosarcoma cells (9), is a general phenomenon, we examined several different cell types. The transient PARP activation occurs prior to the induction of caspase-3-like activity, as measured by the in vitro cleavage of [35S]methionine-labeled PARP into 89- and 24-kDa fragments by cell extracts derived at various stages of apoptosis. This burst of PAR synthesis also occurs before the onset of internucleosomal DNA fragmentation, a biochemical hallmark of apoptosis, which has been shown to begin on day 7 and peak at day 10, when almost 100% of the cells have undergone apoptosis (4, 9). Although caspase-3 is the major protease responsible for the in vivo cleavage of PARP, other caspases have also been shown to partially cleave PARP at much lower efficiencies; thus, we refer to this activity as caspase-3-like.
The time course of apoptosis induced by the topoisomerase inhibitor camptothecin in HL-60 cells is shorter than spontaneous apoptosis occurring in osteosarcoma cells, as evidenced by the detection of internucleosomal DNA fragmentation by 6 h after induction (Fig. 1B). Caspase-3-like PARP-cleavage activity was apparent at 4 h and maximal at 6-8 h after induction (Fig. 1C). Immunoblot analysis with antibodies to PAR revealed an early transient peak of poly(ADP-ribosyl)ation of nuclear proteins 2 h after induction of apoptosis (Fig. 1A), prior to the onset of caspase-3-like activity and DNA fragmentation, similar to that observed in osteosarcoma cells. Caspase-3-like cleavage of PARP apparently results in PARP inactivation as there are no poly(ADP-ribosyl)ated acceptors at the peak of caspase-3 like activity (6-8 h), even in the presence of massive DNA fragments (Fig. 1B) at this time.
|
Transient Poly(ADP-ribosyl)ation of Nuclear Proteins Occurs Early during Fas-mediated Apoptosis in Murine 3T3-L1 Cells but Not in PARP-depleted 3T3-L1 Antisense Cells-- We next examined murine 3T3-L1 cells that can be depleted of endogenous PARP by antisense RNA expression. We have previously used these cells, which are stably transfected with a dexamethasone-inducible PARP antisense construct, to investigate the role of PARP in differentiation (11, 15, 63). PARP was shown to be required for a round of DNA replication that precedes the onset of differentiation in these cells.
Because apoptosis has previously been induced in 3T3-L1 cells only by deregulated expression of c-Myc under conditions of serum deprivation (21), it was first necessary to establish conditions under which apoptosis could be triggered by exogenous inducers. 3T3-L1 cells transfected with the control vector (mock-transfected) were preincubated in the presence or absence of 1 mM dexamethasone for 72 h and then treated with various inducers of apoptosis. Tumor necrosis factor-
|
|
|
Effects of the Absence of Early Transient Poly(ADP-ribosyl)ation on Morphological and Biochemical Markers of Apoptosis in 3T3-L1 Cells-- We next tried to determine whether prevention of the early burst of PAR synthesis by PARP antisense RNA expression could affect the development of other biochemical or morphological markers of apoptosis when these cells are exposed to apoptosis inducers. The combination of anti-Fas and cycloheximide induced a marked increase in caspase-3-like activity in mock-transfected 3T3-L1 cells that had been preincubated in the absence or presence of dexamethasone (Fig. 5A); this effect was maximal 24 h after induction of apoptosis. Whereas PARP-antisense 3T3-L1 cells that were not exposed to dexamethasone showed a similar increase in caspase-3-like activity in response to anti-Fas and cycloheximide, no such increase was apparent in PARP-antisense cells that had been depleted of PARP by preincubation with dexamethasone before exposure to anti-Fas and cycloheximide (Fig. 5B).
|
|
Effects of PARP Depletion by Antisense RNA Expression on Induction of Apoptosis in Human Jurkat Cells-- To confirm our results with 3T3-L1 control and antisense cells, we examined human Jurkat T cells stably transfected with either a PARP antisense RNA construct or the empty vector. Immunoblot analysis showed that preincubation of two different Jurkat antisense cell clones for 72 h with dexamethasone resulted in depletion of endogenous PARP by ~99% (Fig. 7A).
|
Transient Poly(ADP-ribosyl)ation of Nuclear Proteins Also Occurs
Early during Fas-mediated Apoptosis in PARP +/+ Fibroblasts but Not in
PARP /
Cells--
To investigate whether prevention of the early
burst of PAR synthesis by gene disruption could likewise affect the
induction of biochemical or morphological markers of apoptosis when
these cells are exposed to apoptosis inducers, fibroblasts derived from wild-type (PARP +/+) and PARP knockout mice (PARP
/
) (12), immortalized by the standard 3T3 protocol (28), were utilized. PARP
/
cells were confirmed devoid of PARP and PAR by immunoblot analysis with the corresponding antibodies (Fig.
8A). As with the other cell
lines, these cells also exhibited a transient burst of
poly(ADP-ribosyl)ation of nuclear proteins as early as 1 h after
exposure to anti-Fas and cycloheximide (Fig. 8B), and PAR synthesis markedly declined thereafter, presumably by a combination of
caspase-3-like mediated PARP cleavage and PAR-glycohydrolase activity. As anticipated, no burst of poly(ADP-ribosyl)ation was observed in PARP
/
fibroblasts after exposure to inducers of apoptosis for up to 6 h (Fig. 8B).
|
Immortalized Fibroblasts Derived from PARP Knockout Mice Do Not
Exhibit Morphological and Biochemical Markers Characteristic of
Apoptosis--
Anti-Fas and cycloheximide induced a marked increase in
caspase-3-like activity in PARP +/+ cells; this effect was maximal 24 h after induction of apoptosis, as indicated by the complete cleavage of PARP into 89- and 24-kDa fragments (Fig.
9A). In contrast, no such
increase in caspase-3-like activity was evident in PARP /
cells
after exposure to anti-Fas and cycloheximide for up to 24 h.
|
|
Transfection of PARP /
Fibroblasts with Wild-type PARP
Sensitizes These Cells to Fas-mediated Apoptosis--
PARP
/
fibroblasts were stably transfected with pcD-12, a plasmid expressing
wild-type PARP (17). Immunoblot analysis showed that three different
cell clones (1, 2, and 3) and pooled clones (P) expressed PARP protein
similar to the PARP +/+ cells, whereas PARP
/
cells and the clone
transfected with the vector alone ("vec") did not (Fig.
11A). The ability of these
clones to express PARP was also confirmed by in vitro PARP
activity assays (data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To investigate, without the use of possibly nonspecific chemical inhibitors (30-32), the potential roles of PARP and poly(ADP-ribosyl)ation in nuclear processes that require cleavage and rejoining of DNA strands, we have previously established and characterized several mammalian cell lines, including HeLa (16), keratinocytes (33), and 3T3-L1 preadipocytes (11), that are stably transfected with PARP antisense cDNA under the control of an inducible promoter. Establishment of conditions at which endogenous PARP protein and activity can be substantially depleted at specific times by expression of PARP antisense transcripts has enabled us to investigate the roles of PARP in DNA repair, recovery of cells from exposure to mutagenic agents (10, 33), gene amplification (10), and differentiation-linked DNA replication (11, 15, 63).
A role for PARP in apoptosis has been suggested by studies showing that the enzyme undergoes proteolytic cleavage into 89- and 24-kDa fragments during chemotherapy-induced (2) or spontaneous (4) apoptosis. PARP cleavage by caspase-3 has been shown to be necessary for apoptosis (4, 5); the cleavage and inactivation of PARP as well as subsequent apoptotic events are blocked by a peptide inhibitor of this protease. We recently showed by immunofluorescence microscopy that poly(ADP-ribosyl)ation of nuclear proteins occurs early in apoptosis, prior to commitment to cell death, and is followed by cleavage and inactivation of PARP; only small amounts of PAR remained during the later stages of apoptosis, despite the presence of a large number of DNA strand breaks (9). We have now shown that the transient burst of poly(ADP-ribosyl)ation of nuclear proteins during the early stages of apoptosis occurs in several other cell systems as well. Furthermore, by depleting the normally abundant PARP from 3T3-L1 and Jurkat cells by antisense RNA expression prior to the induction of apoptosis, or with the use of immortalized fibroblasts derived from PARP knockout mice, we have demonstrated that prevention of this early activation of PARP blocks various biochemical and morphological changes associated with apoptosis, thus correlating the early poly(ADP-ribosyl)ation with later events in the Fas-mediated cell death cascade.
In contrast, it was recently shown that primary, nonimmortalized PARP
/
splenocytes and fibroblasts, from the same strain of PARP
knockout mice from which the immortalized fibroblasts used in the
present study were derived, undergo apparently normal apoptosis in
response to anti-Fas, TNF-
,
-irradiation, or dexamethasone (14).
Although the concentration of anti-Fas used to induce apoptosis in
these primary PARP
/
cells was substantially higher than that used
in our study with immortalized fibroblasts, the apparent discrepancy
between the responses of the primary and immortalized cells remains to
be clarified. This difference between the two types of cells may be
related to the process of immortalization. The immortalized fibroblasts
used in the present study are essentially clonal in comparison, whereas
the primary cultures used in previous studies (13, 14) contain cells at
various stages of development. Although the physiological relevance of
using immortalized cell lines requires further study, our results with
PARP-antisense cell lines (both murine and human) are consistent with
our data obtained with the immortalized PARP
/
fibroblasts.
In both 3T3-L1 and Jurkat PARP-antisense cells, endogenous PARP protein
was substantially depleted after incubation with 1 µM
dexamethasone for 72 h, whereas PARP abundance was unaffected by
dexamethasone treatment in control cells. With the use of an in
vitro PARP-cleavage assay, marked induction of caspase-3-like activity was observed after exposure to anti-Fas and cycloheximide in
control 3T3-L1 cells and immortalized PARP +/+ fibroblasts, as well as
in control Jurkat cells treated with anti-Fas, and HL-60 cells exposed
to camptothecin. These various treatments also induced a transient
burst of poly(ADP-ribosyl)ation before the onset of biochemical events
associated with apoptosis in these cells. In contrast, PARP-depleted
3T3-L1 or Jurkat antisense cells as well as immortalized PARP /
fibroblasts did not undergo this early transient poly(ADP-ribosyl)ation
nor did they show any increase in caspase-3-like activity, demonstrate
internucleosomal DNA fragmentation, or exhibit morphological changes
characteristic of apoptosis.
Proteolytic cleavage and processing of CPP32 into mature caspase-3 was
also impaired in 3T3-L1 cells depleted of PARP by antisense RNA
expression and in PARP /
fibroblasts. Various lines of evidence indicate that caspase-3 is both necessary and sufficient to trigger apoptosis: (i) disruption of the caspase-3 gene in knockout mice results in excessive accumulation of neuronal cells due to a lack of
apoptosis in the brain (34); (ii) multiple apoptotic signals, including
serum withdrawal, Fas activation, ionizing radiation, and various
pharmacological agents, activate caspase-3 by proteolytic cleavage of
CPP32 (35-39); (iii) a tetrapeptide inhibitor of caspase-3 (AcDEVD-CHO) blocks initiation of the apoptotic program in response to
various stimuli (4, 40); and (iv) addition of active caspase-3 to
normal cytosol can activate the apoptotic program (41). Caspase-3 is
responsible for the cleavage of various cellular substrates at the
onset of apoptosis, including PARP, U1-70K, DNA-dependent protein kinase, the retinoblastoma, protein (Rb), fodrin, actin, lamin,
gelsolin, and an inhibitor of a caspase-activated DNase (ICAD) (4, 5,
42-47); these proteins are implicated in DNA repair, mRNA
splicing, regulation of the cell cycle, or in morphological changes and
DNA fragmentation associated with apoptosis. However, it is still
unclear whether cleavage of any one substrate is sufficient for a cell
to be committed to apoptosis. The biological significance and
biochemical consequences of PARP cleavage and its consequent inactivation also remain unclear.
The substantial extent of nuclear poly(ADP-ribosyl)ation apparent early during apoptosis in 3T3-L1, HL-60, Jurkat, and osteosarcoma cells as well as in immortalized PARP +/+ fibroblasts is consistent with the appearance of large (1 Mb) chromatin fragments at this reversible stage (6), given that the activity of PARP is absolutely dependent on DNA strand breaks. A marked decrease in NAD concentration, indicative of increased PAR synthesis, and a subsequent recovery in NAD levels prior to the appearance of internucleosomal DNA cleavage have also been previously observed (48). PARP activation has been detected during apoptosis induced by various DNA-damaging agents, including alkylating agents, topoisomerase inhibitors, Adriamycin, x-rays, ultraviolet radiation, mitomycin C, and cisplatin (49-53).
Whether this transient burst of PARP activity plays an important role or is simply a consequence of the presence of large 1-Mb DNA fragments at this early stage of apoptosis remains to be clarified. In the present study, several of the morphological and biochemical markers of apoptosis, such as development of nuclear apoptotic morphology, internucleosomal DNA fragmentation, proteolytic processing and activation of CPP32, and an increase in caspase-3-like activity, did not occur in cells when this early burst of poly(ADP-ribosyl)ation was prevented by either gene disruption or by antisense RNA expression. The early activation of PARP was confirmed by immunofluorescence staining or immunoblot analysis in the various cell lines studied. Thus, PARP activation and poly(ADP-ribosyl)ation of relevant nuclear proteins during the early stages of apoptosis may be required for progression through the death program. Subsequent degradation of PARP may prevent the depletion of NAD and ATP needed for later steps in apoptosis (9).
Previous studies have suggested a correlation between poly(ADP-ribosyl)ation of nuclear proteins and internucleosomal DNA fragmentation during apoptosis, as indicated by a suppressive effect of PARP inhibitors on DNA fragmentation (54, 55) or on nuclear fragmentation (56). However, the relevant target proteins for PARP during apoptosis remain to be identified. Poly(ADP-ribosyl)ation of histone H1, for example, during the early stages of apoptosis was suggested to facilitate internucleosomal DNA fragmentation by enhancing chromatin susceptibility to cellular endonucleases (53).
In preliminary studies (to be published elsewhere), we have obtained
some potentially relevant targets for poly(ADP-ribosyl)ation during the
burst of PAR synthesis at the early stages of apoptosis. These results
show that induction of spontaneous apoptosis in osteosarcoma cells is
associated with an increase in the intracellular abundance of p53.
Immunoprecipitation and immunoblot analysis further indicate that
extensive poly(ADP-ribosyl)ation of p53 occurs concomitant with the
burst of poly(ADP-ribosyl)ation and that subsequent degradation of PAR
attached to p53 occurs concomitant with the increase in caspase-3-like
activity. Thus, this posttranslational modification may play a role in
the regulation of p53 function or, alternatively, in its degradation
during p53-dependent apoptosis. These results are
consistent with recent studies showing substantial poly(ADP-ribosyl)ation of p53, with polymer chain lengths from 4 to 30 residues, in cells undergoing apoptosis in response to DNA damage (57,
58). Electrophoretic mobility shift analysis further showed that
ADP-ribose polymers attached to p53 blocked its sequence-specific
binding to a 26-base pair oligonucleotide containing the palindromic
p53 consensus binding sequence, suggesting that poly(ADP-ribosyl)ation
of p53 may negatively regulate p53-mediated transcriptional activation
of genes important in the cell cycle and apoptosis (59). Recently,
primary fibroblasts from PARP /
mice were further shown to have a
2-fold lower basal level of p53 and are defective in the induction of
p53 in response to DNA damage (60).
Finally, the activity of Ca2+,
Mg2+-dependent endonuclease is inhibited by
poly(ADP-ribosyl)ation in vitro (61), and the enzyme is
implicated in internucleosomal DNA cleavage during apoptosis; it is
identical in size and kinetic properties to DNase , which is thought
to be responsible for DNA fragmentation during thymic apoptosis (62).
This enzyme also seems to be a target of the early burst of
poly(ADP-ribosyl)ation during spontaneous apoptosis in osteosarcoma and
HL-60 cells (data not shown), suggesting a possible negative regulatory
role for PARP in apoptosis, whereby inactivation by caspase-3-catalyzed
cleavage may release specific nuclear proteins from
poly(ADP-ribosyl)ation-induced inhibition. These ongoing studies aim to
clarify the apparently essential requirement, at least in the clonal
immortalized mouse and human cells studied here, for the early and
brief poly(ADP-ribosyl)ation that occurs during the initial stages of
apoptosis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. H. Hilz for the polyclonal antibody to murine PARP, Dr. D. Nicholson for the antibody to CPP32, and Drs. M. Miwa and T. Sugimura for the antibody to PAR. We also thank Drs. S. Spiegel, E. Gelmann, A. Dritschilo, Z.-Q. Wang, and J. Cossman for critical review of the manuscript and K. Brocklehurst for help in editing it.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Cancer Institute Grants CA25344 and CA13195, the United States Air Force Office of Scientific Research Grant AFOSR-89-0053, and the United States Army Medical Research and Development Command Contracts DAMD17-90-C-0053 (to M. E. S.) and DAMD 17-96-C-6065 (to D. S. R.).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.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Georgetown University School of Medicine, Basic
Science Bldg., Rm. 351, 3900 Reservoir Rd., NW, Washington, DC 20007. Tel.: 202-687-1718; Fax: 202-687-7186; E-mail:
smulson{at}bc.georgetown.edu.
1
The abbreviations used are: PARP,
poly(ADP-ribose) polymerase; PAR, poly(ADP-ribose); MNU,
N-methyl-N-nitrosourea; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Pipes,
1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline;
TNF-, tumor necrosis factor-
.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|