Detection of apoptosis induced by topoisomerase inhibitors and serum deprivation in syrian hamster embryo cells

The sensitivity of normal diploid Syrian hamster embryo (SHE) cells to apoptosis was tested after treatment with the topoisomerase inhibitors camptothecin and etoposide and after serum withdrawal. Programmed cell death (PCD) was identified through morphological, biochemical, and molecular changes and compared with that of HL60 cell line. The results showed that topoisomerase inhibitors, which were shown to be potent PCD inducers in the HL60 cell line, induced a weaker apoptotic response in SHE cells than after growth factor deprivation. In addition, serum-free medium, which rapidly induced apoptosis in SHE cells, did not affect the HL60 cell line. In both cell types, PCD was expressed by condensed chromatin, fragmented nuclei, and DNA laddering on electrophoretic gels, an indisputable sign of apoptosis. In apoptotic HL60 cells, the cleavage of 113-kDa poly(ADP-ribose)polymerase (PARP) resulted in the so-called apoptotic 89-kDa fragment and was associated with increased caspase-3 activity. In apoptotic SHE cells, PARP degraded early but the degradation profile was not characterized by the appearance of an 89-kDa fragment. Moreover, no activation of caspase-3 was noted. ZnCl(2), which is known to prevent protease activity responsible for apoptosis features, inhibited PARP cleavage and nuclear modifications induced by apoptotic stimuli in both cell types, but with a higher sensitivity in SHE cells. Apoptosis induced by serum deprivation was linked with c-myc negative regulation in SHE cells, but not with p53 protein accumulation, while topoisomerase inhibitors led to p53 stabilization without any change in c-myc expression. Serum-free medium and topoisomerase inhibitors did not modify c-myc expression in the HL60 cell line. The overall results demonstrated that apoptosis, which is a carefully regulated process of cell death, may proceed through mechanisms varying according to cell type or apoptosis inducer. In addition, markers which are generally considered hallmarks of apoptosis may fail to appear in some cell types.     

Cleavage of Poly(ADP-ribose) polymerase measured in situ in individual cells: relationship to DNA fragmentation and cell cycle position during apoptosis

Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a target of caspases during apoptosis: its cleavage onto 89- and 24-kDa fragments is considered to be a hallmark of the apoptotic mode of cell death. Another hallmark is the activation of endonuclease which targets internucleosomal DNA. The aim of the present study was to reveal cell cycle phase specificity as well as the temporal and sequence relationships of PARP cleavage vis-à-vis DNA fragmentation in two model systems of apoptosis, one induced by DNA damage via cell treatment with camptothecin (CPT) (mitochondria-induced pathway) and another by the cytotoxic ligand tumor necrosis factor alpha (TNF-alpha) (cell surface death receptor pathway). PARP cleavage was detected immunocytochemically using antibody which recognizes its 89-kDa fragment (PARP p89) while DNA fragmentation was assayed by in situ labeling of DNA strand breaks. The frequency and extent of PARP cleavage as well as DNA fragmentation were measured by mutiparameter flow and laser scanning cytometry. PARP cleavage, selective to S phase cells, was detected 90 min after administration of CPT. PARP cleavage in the cells treated with TNF-alpha was not selective to any cell cycle phase and was seen already after 30 min. DNA fragmentation trailed PARP cleavage by about 30 min and showed a similar pattern of cell cycle specificity. PARP p89 was present in nuclear chromatin but at least in the early phase of apoptosis it did not colocalize with DNA strand breaks. The rate of cleavage of PARP molecules in individual cells whether induced by CPT or TNF-alpha was rapid as reflected by the paucity of cells with a mixture of cleaved and noncleaved PARP molecules. In contrast, DNA fragmentation proceeded stepwise before reaching the maximal number of DNA strand breaks. Although time windows for PARP cleavage vs DNA fragmentation were different at early stages of apoptosis, a good overall correlation between the cytometric assays of apoptotic cells identification based on these events was observed in both CPT- and TNF-alpha-treated cultures.     

Selective loss of poly(ADP-ribose) and the 85-kDa fragment of poly(ADP-ribose) polymerase in nucleoli during alkylation-induced apoptosis of HeLa cells.

Alkylation treatment of HeLa cells results in the rapid induction of apoptosis as revealed by DNA laddering and cleavage of poly(ADP-ribose) polymerase (PARP) into the 29-and 85-kDa fragments (Kumari S. R., Mendoza-Alvarez, H. & Alvarez-Gonzalez, R. (1998) Cancer Res. 58, 5075-5078). Here, we performed a time-course analysis of (i) poly(ADP-ribose) synthesis and degradation as well as (ii) the subnuclear localization of PARP and its fragments by using confocal laser scanning immunofluorescence microscopy. PARP was activated within 15 min post-treatment, as revealed by nuclear immunostaining with antibody 10H (recognizing poly(ADP-ribose)). This was followed by a late, time-dependent, progressive decline of 10H signals that coincide with the time of PARP cleavage. Strikingly, nucleolar immunostaining with antibodies 10H and C-II-10 (recognizing the 85-kDa PARP fragment) was lost by 15 min post-treatment, whereas F-I-23 signals (recognizing the 29-kDa fragment) persisted. We hypothesize that the 85-kDa PARP fragment is translocated, along with covalently bound poly(ADP-ribose), from nucleoli to the nucleoplasm, whereas the 29-kDa fragment is retained, because it binds to DNA strand breaks. Our data (i) provide a link between the known time-dependent bifunctional role of PARP in apoptosis and the subcellular localization of PARP fragments and also (ii) add to the evidence for early proteolytic changes in nucleoli during apoptosis.     

Poly (ADP-ribose) polymerase cleavage monitored in situ in apoptotic cells

During apoptosis, the activation of a family of cysteine proteases, or caspases, results in proteolytic cleavage of numerous substrates. Antibody probes specific for neoepitopes on protein fragments generated by caspase cleavage provide a means to monitor caspase activity at the level of the individual cell. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a well-known substrate for caspase-3 cleavage during apoptosis. Its cleavage is considered to be a hallmark of apoptosis. Here, we demonstrate that an affinity-purified polyclonal antibody to the p85 fragment of PARP is specific for apoptotic cells. Western blots show that the antibody recognizes the 85-kDa (p85) fragment of PARP but not full-length PARP. We demonstrate a time course of PARP cleavage and DNA fragmentation in situ using the PARP p85 fragment antibody and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in Jurkat cells treated with anti-Fas. Furthermore, our results indicate that the p85 fragment of PARP resulting from caspase cleavage during apoptosis is rapidly localized outside the condensed chromatin but not in the cytoplasm.     

Apparent cleavage of poly(ADP-ribose) polymerase in non-apoptotic mouse LTA cells: An artifact of cross-reactive secondary antibody

Proteolytic cleavage of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) to fragments of 89 kD and 24 kD is widely observed during apoptotic cell death. In the present study, labelling of a M_r 89000 polypeptide was demonstrated in untreated mouse LTA cells during probing of immunoblots with C-2-10 monoclonal anti-PARP antibody. The source of the labeling was traced to the secondary antibody preparation, which labeled a M_r ~89000 polypeptide in murine LTA cells but not in human cells. These observations indicate that assessment of PARP cleavage must be (1) performed with appropriate controls when new cell lines are investigated and (2) carefully interpreted in light of additional biochemical or morphological data demonstrating apoptotic changes.     

Involvement of poly(ADP-ribose) polymerase and activation of caspase-3-like protease in heat shock-induced apoptosis in tobacco suspension cells

The cleavage of poly(ADP-ribose) polymerase (PARP) by caspase (casp)-3 is an essential link in the apoptotic pathway in animal cells. In plant cells, however, there is no authentic evidence for the similar role that PARP may play during apoptosis. Using a heat shock (HS)-induced apoptosis system of tobacco cells, we found that immediately after a 4 h heat treatment, PARP was cleaved to form an 89 kDa signature fragment, while DNA laddering appeared only after a 20 h recovery following the HS. An activation of casp-3-like protease was also observed. The results suggest that apoptosis in plants and animals may share common mechanisms. On the other hand, when cells were preincubated with 4 mM 3-aminobenzamide or 2-8 mM nicotinamide, the specific inhibitors of PARP, before HS treatment, apoptotic cell death was reduced significantly. Our results thus imply that PARP may also be involved in apoptosis in a different way from the casp-related events.     

Effect of staurosporine-induced apoptosis on endothelial nitric oxide synthase in transfected COS-7 cells and primary endothelial cells

Nitric oxide (NO) may block apoptosis by inhibiting caspases via S-nitrosylation of cysteines. Here, we investigated whether effector caspases might cleave and thereby inhibit endothelial nitric oxide synthase (eNOS). Exposure of eNOS-transfected COS-7 cells and bovine aortic endothelial cells to staurosporine resulted in significant loss of 135-kDa eNOS protein and activity, and appearance of a 60-kDa eNOS fragment; effects were inhibited by the general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp[OMe]-fluoromethyl ketone (zVAD-fmk). In eNOS-transfected COS-7 cells, staurosporine-induced activation of caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage coincided with increased eNOS degradation and decreased activity. Loss of eNOS activity was greater than the degree of proteolysis. Incubation of immunoprecipitated eNOS with caspase-3, caspase-6 or caspase-7 resulted in eNOS cleavage. Staurosporine, a general protein kinase inhibitor, also reduced phosphorylation and decreased calmodulin binding, an effect that may explain the reduction in activity. eNOS, therefore, is both an inhibitor of apoptosis and a target of apoptosis-associated proteolysis.     

Activation of caspase 3 in HL-60 cells exposed to hydrogen peroxide

Recent studies have suggested that hydrogen peroxide (H2O2), a reactive compound formed endogenously in the breakdown of superoxide, may mediate the induction of apoptosis in various cell types in response to external stimuli. However, the role of H2O2 in the apoptotic pathway has not been clearly established. The purpose of this study was to determine if H2O2 treatment could induce apoptosis through the activation of caspases. Doses of H2O2 ranging from 10 microM to 100 microM, when added to HL-60 cells, resulted in the cleavage of poly(ADP-ribose) polymerase (PARP) from its native 113 Kd form to a processed 89 Kd fragment, indicative of cells undergoing apoptosis. PARP was predominantly in the fragmented form when doses of 20 microM and greater were used. A time course study of changes in PARP processing in H2O2-treated cells revealed that 10 and 50 microM H2O2 required 6 and 3 h, respectively, to specifically degrade PARP, suggesting that the H2O2-induced PARP cleavage is both time and concentration dependent. Since PARP is cleaved by CPP32 (caspase-3), we next determined if H2O2 was capable of effecting changes in CPP32 activity. The caspase activity was assayed using a colorimetric substrate, DEVD-pNa. Results of these experiments showed that H2O2 increased caspase activity at 3 h, corresponding to the time of appearance of fragmented PARP. Also, CPP32 activity and PARP processing were both significantly suppressed by caspase-3 inhibitors. Taken together, these results suggest that H2O2 mediates specific cleavage of PARP and possibly apoptosis by activating caspase 3.     

Inhibition of homodimerization of poly(ADP-ribose) polymerase by its C-terminal cleavage products produced during apoptosis

The biochemical role of the C-terminal fragment of poly(ADP-ribose) polymerase (PARP) was investigated in HeLa cells undergoing UV-mediated apoptosis. During the course of apoptosis, the C-terminal cleavage product of PARP interacted with intact PARP and down-regulated PARP activity by blocking the homodimerization of PARP. The basic leucine zipper motif in the auto-modification domain of the C-terminal fragment of PARP represented the site of association, and Leu(405) was critical to the ability of the basic leucine zipper motif to associate with intact PARP. The expression of the C-terminal fragment of PARP stimulated UV-mediated apoptosis. These results suggest that the C-terminal cleavage product of PARP produced during apoptosis blocks the homodimerization of PARP and inhibits the cellular PARP activity. The inhibition of the cellular PARP activity might prevent cellular NAD(+) depletion and stimulate apoptosis by maintaining the basal cellular energy level required for the completion of apoptosis.     

Detection of DNA breaks in apoptotic cells utilizing the DNA binding domain of poly(ADP-ribose) polymerase with fluorescence microscopy.

The DNA binding domain (DBD) of poly(ADP-ribose) polymerase (PARP) has proved to be a novel, highly sensitive probe for detecting DNA breaks in intact cells undergoing apoptosis. A recombinant peptide spanning the DNA binding domain of PARP was expressed, purified and used to detect DNA strand breaks in fixed cells. Fluorescence microscopy with this probe followed by detection with anti-PARP antisera initially revealed an increased binding following treatment of cells with DNA strand-breaking agents (such asN-methyl-N'-nitro-N-nitrosoguanidine) and, subsequently, using biotinylated PARP DBD, during the later stages of apoptosis in several cell systems, when internucleosomal strand breaks became evident. This procedure was found to be at least as sensitive and required fewer steps to detect DNA strand breaks than those utilizing Klenow incorporation of biotinylated nucleotides.     

Activation of a cysteine protease in MCF-7 and T47D breast cancer cells during beta-lapachone-mediated apoptosis

beta-Lapachone (beta-lap) effectively killed MCF-7 and T47D cell lines via apoptosis in a cell-cycle-independent manner. However, the mechanism by which this compound activated downstream proteolytic execution processes were studied. At low concentrations, beta-lap activated the caspase-mediated pathway, similar to the topoisomerase I poison, topotecan; apoptotic reactions caused by both agents at these doses were inhibited by zVAD-fmk. However at higher doses of beta-lap, a novel non-caspase-mediated "atypical" cleavage of PARP (i.e., an approximately 60-kDa cleavage fragment) was observed. Atypical PARP cleavage directly correlated with apoptosis in MCF-7 cells and was inhibited by the global cysteine protease inhibitors iodoacetamide and N-ethylmaleimide. This cleavage was insensitive to inhibitors of caspases, granzyme B, cathepsins B and L, trypsin, and chymotrypsin-like proteases. The protease responsible appears to be calcium-dependent and the concomitant cleavage of PARP and p53 was consistent with a beta-lap-mediated activation of calpain. beta-Lap exposure also stimulated the cleavage of lamin B, a putative caspase 6 substrate. Reexpression of procaspase-3 into caspase-3-null MCF-7 cells did not affect this atypical PARP proteolytic pathway. These findings demonstrate that beta-lap kills cells through the cell-cycle-independent activation of a noncaspase proteolytic pathway.     

Caspase-dependent apoptosis and -independent poly(ADP-ribose) polymerase cleavage induced by transforming growth factor beta1

Apoptosis is an important cell suicide program which involves the caspases activation and is implicated in physiological and pathological processes. Poly(ADP-ribose) polymerase (PARP) cleavage is often associated with apoptosis and has been served as one hallmark of apoptosis and caspase activation. In this study, we aimed to determine TGF-beta1-induced apoptosis and to examine the involvement of caspases and its relationship with PARP cleavage. TGF-beta1 induces strong apoptosis of AML-12 cells which can be detected by DNA fragmentation, FACS, and morphological assays. Z-VAD-fmk, a selective caspase inhibitor, partially inhibits the TGF-beta1-induced apoptosis; but has no effect on TGF-beta1-induced DNA fragmentation and PARP cleavage. However, BD-fmk, a broad-spectrum caspase inhibitor, completely suppresses TGF-beta1-induced apoptosis, but unexpectedly does not inhibit TGF-beta1-induced PARP cleavage. Furthermore, Z-VAD-fmk treatment is able to completely inhibit the daunorubicin-induced apoptosis in A-431 cells, but only slightly blocks the daunorubicin-induced PARP cleavage, whereas BD-fmk can inhibit both daunorubicin-induced apoptosis and PARP cleavage completely. In addition, we observed that both TGF-beta1-induced apoptosis and PARP degradation in AML-12 cells can be completely blocked by inhibiting the protein synthesis with cycloheximide. These results demonstrate for the first time that TGF-beta1-induced caspase-dependent apoptosis is associated with caspase-independent PARP cleavage that requires the TGF-beta1-induced synthesis of new proteins. The results indicate that caspase-3 is not a major caspase involved in TGF-beta1-induced apoptosis in AML-12 cells, and is not required for apoptosis-associated DNA fragmentation. The results also suggest that PARP cleavage may occur as an independent event that can be disassociated with cell apoptosis.     

In vivo and in vitro activation of caspase-8 and -3 associated with Helicobacter pylori infection

In vivo and in vitro studies have shown an increase in apoptosis in gastric epithelial cells in persons infected with Helicobacter pylori. H. pylori-induced activation of caspase-8 and -3 was evaluated using a human gastric adenocarcinoma cell line (AGS) and gastric tissue from humans and monkeys colonized with H. pylori. The enzymatic activity of caspase-8 was detected only in AGS cells exposed to H. pylori up to 24 h. The active form of caspase-8 was present by Western blot after exposure to H. pylori for 3 h and persisted through 24 h. Caspase-3 activity was present in AGS cells exposed to H. pylori for 3 h, reaching a maximum after 24 h (a sevenfold increase in activity). Caspase-8-mediated cleavage of procaspase-3 generated a 20-kDa band (indicative of the presence of active caspase-3) present only in AGS cells exposed to H. pylori. Active caspase-3 staining was markedly increased in gastric mucosa from infected persons and animals, compared to uninfected controls by immunohistochemistry. Stimulation of downstream events leading to apoptosis, such as the cleavage of PARP (poly adenosine-diphosphate-ribose polymerase) and DFF45 (DNA fragmentation factor 45) as a result of activation of caspase-3, was evaluated. PARP was cleaved, resulting in the presence of both an 89- and a 24-kDa band along with DFF45, resulting in the presence of 10- and 12-kDa bands only in gastric cells exposed to H. pylori. Our data show that H. pylori stimulates the activation of caspases and downstream mediators of caspase-induced apoptosis. This suggests that H. pylori-induced apoptosis is mediated through caspase pathways, which include the activation of caspase-8 and subsequent cleavage and activation of caspase-3. This is consistent with caspase-3 activation that was found in the gastric mucosa of humans and monkeys infected with H. pylori.     

A serine protease is involved in the initiation of DNA damage-induced apoptosis

Caspases are considered to be the key effector proteases of apoptosis. Initiator caspases cleave and activate downstream executioner caspases, which are responsible for the degradation of numerous cellular substrates. We studied the role of caspases in apoptotic cell death of a human melanoma cell line. Surprisingly, the pancaspase inhibitor zVAD-fmk was unable to block cleavage of poly(ADP-ribose) polymerase (PARP) after treatment with etoposide, while it did prevent DEVDase activity. It is highly unlikely that caspase-2, which is a relatively zVAD-fmk-resistant caspase, is mediating etoposide-induced PARP cleavage, as a preferred inhibitor of this caspase could not prevent cleavage. In contrast, caspase activation and PARP degradation were blocked by pretreatment of the cells with the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). We therefore conclude that a serine protease regulates an alternative initiation mechanism that leads to caspase activation and PARP cleavage. More importantly, while zVAD-fmk could not rescue melanoma cells from etoposide-induced death, the combination with AEBSF resulted in substantial protection. This indicates that this novel pathway fulfills a critical role in the execution of etoposide-induced programmed cell death.     

beta-Lapachone-induced apoptosis in human prostate cancer cells: involvement of NQO1/xip3.

beta-Lapachone (beta-lap) induces apoptosis in various cancer cells, and its intracellular target has recently been elucidated in breast cancer cells. Here we show that NAD(P)H:quinone oxidoreductase (NQO1/xip3) expression in human prostate cancer cells is a key determinant for apoptosis and lethality after beta-lap exposures. beta-Lap-treated, NQO1-deficient LNCaP cells were significantly more resistant to apoptosis than NQO1-expressing DU-145 or PC-3 cells after drug exposures. Formation of an atypical 60-kDa PARP cleavage fragment in DU-145 or PC-3 cells was observed after 10 microM beta-lap treatment and correlated with apoptosis. In contrast, LNCaP cells required 25 microM beta-lap to induce similar responses. Atypical PARP cleavage in beta-lap-treated cells was not affected by 100 microM zVAD-fmk; however, coadministration of dicoumarol, a specific inhibitor of NQO1, reduced beta-lap-mediated cytotoxicity, apoptosis, and atypical PARP cleavage in NQO1-expressing cells. Dicoumarol did not affect the more beta-lap-resistant LNCaP cells. Stable transfection of LNCaP cells with NQO1 increased their sensitivity to beta-lap, enhancing apoptosis compared to parental LNCaP cells or vector-alone transfectants. Dicoumarol increased survival of beta-lap-treated NQO1-expressing LNCaP transfectants. NQO1 activity, therefore, is a key determinant of beta-lap-mediated apoptosis and cytotoxicity in prostate cancer cells.     

Failure of Poly(ADP-Ribose) Polymerase Cleavage by Caspases Leads to Induction of Necrosis and Enhanced Apoptosis

Activation of poly(ADP-ribose) polymerase (PARP) by DNA breaks catalyzes poly(ADP-ribosyl)ation and results in depletion of NAD+ and ATP, which is thought to induce necrosis. Proteolytic cleavage of PARP by caspases is a hallmark of apoptosis. To investigate whether PARP cleavage plays a role in apoptosis and in the decision of cells to undergo apoptosis or necrosis, we introduced a point mutation into the cleavage site (DEVD) of PARP that renders the protein resistant to caspase cleavage in vitro and in vivo. Here, we show that after treatment with tumor necrosis factor alpha, fibroblasts expressing this caspase-resistant PARP exhibited an accelerated cell death. This enhanced cell death is attributable to the induction of necrosis and an increased apoptosis and was coupled with depletion of NAD+ and ATP that occurred only in cells expressing caspase-resistant PARP. The PARP inhibitor 3-aminobenzamide prevented the NAD+ drop and concomitantly inhibited necrosis and the elevated apoptosis. These data indicate that this accelerated cell death is due to NAD+ depletion, a mechanism known to kill various cell types, caused by activation of uncleaved PARP after DNA fragmentation. The present study demonstrates that PARP cleavage prevents induction of necrosis during apoptosis and ensures appropriate execution of caspase-mediated programmed cell death.     

Two-color fluorescence detection of Poly (ADP-Ribose) Polymerase-1 (PARP-1) cleavage and DNA strand breaks in etoposide-induced apoptotic cells

During apoptosis, the nuclear enzyme Poly(ADP-Ribose) Polymerase-1 (PARP-1) catalyzes the rapid and transient synthesis of poly(ADP-ribose) from NAD+ and becomes inactive when cleaved by caspases. The regulation of these two opposite roles of PARP-1 is still unknown. We have recently investigated PARP-1 activation/degradation in Hep-2 cells driven to apoptosis by actinomycin D. In the present work, we have extended our analysis to the effect of the DNA damaging agent etoposide, and paid attention to the relationship between PARP-1 cleavage and DNA fragmentation. An original fluorescent procedure was developed to simultaneously identify in situ the p89 proteolytic fragment of PARP-1 (by immunolabeling) and DNA degradation (by the TUNEL assay). The presence of p89 was observed both in cells with advanced signs of apoptosis (where the PARP-1 fragment is extruded from the nucleus into the cytoplasm) and in TUNEL-negative cells, with only incipient signs of chromatin condensation; this evidence indicates that PARP-1 degradation in etoposide-treated apoptotic cells may precede DNA cleavage.     

Intact Cell Evidence for the Early Synthesis, and Subsequent Late Apopain-Mediated Suppression, of Poly(ADP-ribose) during Apoptosis

Poly(ADP-ribose) polymerase (PARP), which is catalytically activated by DNA strand breaks, has been implicated in apoptosis, or programmed cell death. A protease (CPP32) responsible for the cleavage of PARP and necessary for apoptosis was recently purified and characterized. The coordinated sequence of events related to PARP activation and cleavage in apoptosis has now been examined in individual cells. Apoptosis was studied in a human osteosarcoma cell line that undergoes a slow (8 to 10 days), spontaneous, and reproducible death program in culture. Changes in the abundance of intact PARP, poly(ADP-ribose) (PAR), and a proteolytic cleavage product of PARP that contains the DNA-binding domain were examined during apoptosis in the context of individual, whole cells by immunofluorescence with specific antibodies. The synthesis of PAR from NAD increased early, within 2 days of cell plating for apoptosis, prior to the appearance of internucleosomal DNA cleavage and before the cells become irreversibly committed to apoptosis, since replating yields viable, nonapoptotic cells. Strong expression of full-length PARP was also detected, by immunofluorescence as well as by Western analysis, during this same time period. However, after ∼4 days in culture, the abundance of both full-length PARP and PAR decreased markedly. After 6 days, a proteolytic cleavage product containing the DNA-binding domain of PARP was detected immunocytochemically and confirmed by Western analysis, both in the nuclei and in the cytoplasm of cells. A recombinant peptide spanning the DNA-binding domain of PARP was expressed, purified, and biotinylated, and was then used as a probe for DNA strand breaks. Fluorescence microscopy with this probe revealed extensive DNA fragmentation during the later stages of apoptosis. This is the first report, using individual,intact cells,demonstrating that poly(ADP-ribosyl)ation of nuclear proteins occurs prior to the commitment to apoptosis, that inactivation and cleavage of PARP begin shortly thereafter, and that very little PAR per se is present during the later stages of apoptosis, despite the presence of a very large number of DNA strand breaks. These results suggest a negative regulatory role for PARP during apoptosis, which in turn may reflect the requirement for adequate NAD and ATP during the later stages of programmed cell death.