Activation of CDC 25 phosphatase and CDC 2 kinase involved in GL331-induced apoptosis

CDC 25 is a dual phosphatase responsible for dephosphorylation and, thus, activation of CDC 2 kinase in G2. Abnormal activation of cyclin B-associated CDC 2 kinase has been implicated in apoptosis induced by cancer chemotherapeutic agents such as paclitaxel (Taxol) and etoposide (VP-16). In this study, we found that the CDC 2 kinase could be transiently activated when nasopharyngeal carcinoma NPC-TW01 cells were treated for 3 h with a new anticancer agent, GL331. GL331 treatment also induced a concomitant increase in CDC 25A phosphatase activity and a reduced level of Tyr-15-phosphorylated CDC 2 in NPC-TW01 cells. Furthermore, subsequent apoptotic DNA fragmentation induced by GL331 could be interrupted by treatment of the cells with the cyclin B1-specific antisense oligonucleotides, suggesting that abnormal activation of cyclin B1-associated CDC 2 kinase and CDC 25A phosphatase was involved in GL331-induced apoptosis. Raf-1 has been shown to associate with CDC 25A and, thus, to stimulate its phosphatase activity. Our results revealed that GL331 could facilitate the association of CDC 25A with Raf-1, resulting in the cascade of CDC 25A phosphatase activation and CDC 2 kinase activation, as well as related signaling pathways, and ultimately causing apoptosis in cancer cells.     

Stimulation of the DNA-dependent protein kinase by poly(ADP-ribose) polymerase

The DNA-dependent protein kinase (DNA-PK) is a heterotrimeric enzyme that binds to double-stranded DNA and is required for the rejoining of double-stranded DNA breaks in mammalian cells. It has been proposed that DNA-PK functions in this DNA repair pathway by binding to the ends of broken DNA molecules and phosphorylating proteins that bind to the damaged DNA ends. Another enzyme that binds to DNA strand breaks and may also function in the cellular response to DNA damage is the poly(ADP-ribose) polymerase (PARP). Here, we show that PARP can be phosphorylated by purified DNA-PK, and the catalytic subunit of DNA-PK is ADP-ribosylated by PARP. The protein kinase activity of DNA-PK can be stimulated by PARP in the presence of NAD+ in a reaction that is blocked by the PARP inhibitor 1, 5-dihydroxyisoquinoline. The stimulation of DNA-PK by PARP-mediated protein ADP-ribosylation occurs independent of the Ku70/80 complex. Taken together, these results show that PARP can modify the activity of DNA-PK in vitro and suggest that these enzymes may function coordinately in vivo in response to DNA damage.     

In situ staining for poly(ADP-ribose) polymerase activity using an NAD analogue

Poly(ADP-ribose) polymerase (PARP) is a highly abundant nuclear enzyme which metabolizes NAD, in response to DNA strand breakage, to produce chains of poly(ADP-ribose) attached to nuclear proteins. PARP activation has been implicated in ischemia/reperfusion injury, but its biological significance is not fully understood. We have modified an existing in situ method for detection of PARP activity by using an NAD analogue in which adenine is modified by an "etheno" (vinyl) bridge. Etheno-NAD serves as a PARP substrate in an initial enzymatic reaction; a specific antibody to ethenoadenosine is then used in an immunohistochemical reaction to detect the production of modified poly(ADP-ribose). The method produces strong and specific labeling of nuclei in which PARP has been activated, i.e., those in which DNA strand breaks have been produced, and the results can be analyzed by microscopy, flow cytometry, or colorimetry. The method is applicable to cultured cells in several formats and to frozen tissue sections. The particular characteristics of the new method may assist in future in situ studies of PARP activation.     

Transient poly(ADP-ribosyl)ation of nuclear proteins and role of poly(ADP-ribose) polymerase in the early stages of apoptosis

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.     

Poly (ADP-ribose) polymerase, a potential target for drugs: Part II. Regulation of differentiation by the poly ADP-ribose system

It is shown that eukaryotic differentiation is specifically sensitive to pADPRT regulation in Trypanosomna, Leishmania and Mytilus models. There is powerful inhibition of early differentiation without cell toxicity by pADPRT ligands.     

Effect of a poly(ADP-ribose) polymerase inhibitor on DNA breakage and cytotoxicity induced by hydrogen peroxide and gamma-radiation

Thirty-six outpatients aged 20 to 51 with RDC primary major depressive disorder (MDD) completed a 5-week trial of desipramine following a week of single-blind placebo. Five had a past history of hypomanic disorder. For all but one patient, daily dosage at bedtime was constant for the final 4 weeks, with a mean (S.D.) of 168.1 (46.5) mg. Plasma samples drawn at the three final weekly visits were assayed by high-performance liquid chromatography for 2-hydroxydesipramine (2-OH-DMI) and desipramine. Mean (S.D.) plasma levels were 59.8 (30.0) ng/ml for 2-OH-DMI and 142.9 (138.6) ng/ml for desipramine. Thirteen patients (36%) had a final 17-item Hamilton depression rating < and = 6 and were classified as responders. According to receiver operating characteristics analysis, patients with plasma 2-OH-DMI levels > and = 58 and < 92 ng/ml had a greater likelihood of responding than those with lower or higher levels (p = 0.005, Fisher's exact test), while patients with plasma desipramine levels > and = 64 ng/ml were more likely to respond than those with lower levels (p = 0.032, Fisher's exact test). Results using an alternate response criterion were similar. These findings suggest that in desipramine-treated outpatients with primary MDD the relationship between therapeutic response and plasma levels is curvilinear for 2-OH-DMI and linear for desipramine.     

Modulation of (+/-)-anti-BPDE mediated p53 accumulation by inhibitors of protein kinase C and poly(ADP-ribose) polymerase

The rapid accumulation of the p53 gene product is considered to be an important component of the cellular response to a variety of genotoxins. In order to gain insights on the biochemical pathways leading to p53 stabilization, the effect of (+/-) 7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydrobenzo(a)-pyrene [(+/-)-anti-BPDE] induced DNA damage on p53 protein levels was investigated in various repair-proficient and repair-deficient human cells. Brief exposure of normal human fibroblasts to 0.05-1 microM (+/-)-anti-BPDE resulted in elevated p53 protein levels as compared to the constitutive levels of control cells. The rapid induction response, detectable within a few hours, was sustained up to a period of at least 24 h. Repair-proficient and repair-deficient (XPA) human lymphoblastoid cells showed a similar response. The poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide (3-AB), diminished the p53 induction response by concomitantly decreasing the extent of (+/-)-anti-BPDE induced DNA damage in cells pretreated with the inhibitor. However, the direct involvement of poly ADP-ribosylation was also apparent as 3-AB was able to attenuate (approximately 50%) the p53 response by post-damage inhibitor treatment of the cells. Inhibition of cellular DNA replication by hydroxyurea and AraC, in the presence or absence of DNA damage, also resulted in rapid p53 accumulation in repair-deficient cells. On the contrary, inhibition of protein kinase C (PKC) by calphostin-C led to an abrogation of (+/-)-anti-BPDE mediated p53 induction. Analysis of the downstream effects of carcinogen treatment showed that the lymphoblastoid cells undergo DNA fragmentation indicative of apoptosis while fibroblasts exhibit cell cycle arrest at the G1-S boundary.     

Induction of hepatic poly(ADP-ribose) polymerase by peroxisome proliferators, non-genotoxic hepatocarcinogens

The binding capacity of FK506 binding protein (FKBP) was examined after 2-h hemispheric ischemia in the gerbil brain in order to clarify the precise mechanism of the neuroprotective effects of FK506. Firstly, the FK506 binding was evaluated in vitro in the normal gerbil brain using 1 nM [3H]dihydro-FK506 as a specific ligand. FK506 binding sites were distributed in a rather homogeneous manner, although the greatest binding was noted in the hippocampus CA1. Secondly, Scatchard analysis demonstrated that the binding sites of FK506 could be composed of two components in each brain region. Thirdly, 18 Mongolian gerbils were divided into two groups: an ischemia group (n = 12) and a sham group (n = 6). The right common carotid artery was ligated to induce hemispheric ischemia for 2 h in the ischemia group. The local cerebral blood flow was measured at the end of the experiment by the [14C]iodoantipyrine method. The ligated animals with levels of local cerebral blood flow in the lateral nuclei of the thalamus of less than 50 ml/100 g/min were utilized as the ischemia group (n=6) for further data analysis. No significant differences in FK506 binding between the ischemia and sham groups were observed in any regions. The above data indicate that the binding capacity of FKBP tends to remain normal during 2-h ischemia, suggesting that FK506 may exert its neuroprotective effects through its binding to FKBP in the brain during the early phase of cerebral ischemia.     

Transcriptional regulation of the rat poly(ADP-ribose) polymerase gene by Sp1

BACKGROUND: Exogenous surfactant therapy of lung donors improves the preservation of normal canine grafts. The current study was designed to determine whether exogenous surfactant can mitigate the damage in lung grafts induced by mechanical ventilation before procurement. METHODS AND RESULTS: Five donor dogs were subjected to 8 hours of mechanical ventilation (tidal volume 45 ml/kg). This produced a significant decrease in oxygen tension (p = 0.007) and significant increases in bronchoscopic lavage fluid neutrophil count (p = 0.05), protein concentration (p = 0.002), and the ratio of poorly functioning small surfactant aggregates to superiorly functioning large aggregates (p = 0.02). Five other animals given instilled bovine lipid extract surfactant and undergoing mechanical ventilation in the same manner demonstrated no significant change in oxygen tension values, lavage fluid protein concentration, or the ratio of small to large aggregates. All 10 lung grafts were then stored for 17 hours at 4 degrees C. Left lungs were transplanted and reperfused for 6 hours. After 6 hours of reperfusion the ratio of oxygen tension to inspired oxygen fraction was 307 +/- 63 mm Hg in lung grafts administered surfactant versus 73 +/- 14 mm Hg in untreated grafts (p = 0.007). Furthermore, peak inspired pressure was significantly (p < 0.05) lower in treated animals from 90 to 360 minutes of reperfusion. Analysis of lavage fluid of transplanted grafts after reperfusion revealed small to large aggregate ratios of 0.17 +/- 0.04 and 0.77 +/- 0.17 in treated versus untreated grafts, respectively (p = 0.009). CONCLUSIONS: Instillation of surfactant before mechanical ventilation reduced protein leak, maintained a low surfactant small to large aggregate ratio, and prevented a decrease of oxygen tension in donor animals. After transplantation, surfactant-treated grafts had superior oxygen tension values and a higher proportion of superiorly functioning surfactant aggregate forms in the air space than untreated grafts. Exogenous surfactant therapy can protect lung grafts from ventilation-induced injury and may offer a promising means to expand the donor pool.     

Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant

We have studied the apoptotic response of poly(ADP-ribose) polymerase (PARP)-/- cells to different inducers and the consequences of the expression of an uncleavable mutant of PARP on the apoptotic process. The absence of PARP drastically increases the sensitivity of primary bone marrow PARP-/- cells to apoptosis induced by an alkylating agent but not by a topoisomerase I inhibitor CPT-11 or by interleukin-3 removal. cDNA of wild type or of an uncleavable PARP mutant (D214A-PARP) has been introduced into PARP-/- fibroblasts, which were exposed to anti-CD95 or an alkylating agent to induce apoptosis. The expression of D214A-PARP results in a significant delay of cell death upon CD95 stimulation. Morphological analysis shows a retarded cell shrinkage and nuclear condensation. Upon treatment with an alkylating agent, expression of wild-type PARP cDNA into PARP-deficient mouse embryonic fibroblasts results in the restoration of the cell viability, and the D214A-PARP mutant had no further effect on cell recovery. In conclusion, PARP-/- cells are extremely sensitive to apoptosis induced by triggers (like alkylating agents), which activates the base excision repair pathway of DNA, and the cleavage of PARP during apoptosis facilitates cellular disassembly and ensures the completion and irreversibility of the process.     

Bcl-2 prevents apoptosis induced by perforin and granzyme B, but not that mediated by whole cytotoxic lymphocytes

Two pathways have been implicated in the induction of apoptosis by cytotoxic T cells: the granule exocytosis pathway and a pathway using CD95 (Fas/APO-1). To test whether apoptosis induced by either of these pathways could be blocked by Bcl-2, we exposed bcl-2-transfected cells to CTL derived from normal, perforin-deficient, or CD95 ligand mutant (gld) mice. Although the levels of Bcl-2 expression achieved were able to protect FDC-P1 and Yac-1 transfectants from a variety of apoptotic stimuli, the cells were not protected from cytolysis mediated by CTL from any of these sources, by NK cells, or granules isolated from CTL. However, Bcl-2 expression significantly inhibited apoptosis induced by purified granzyme B and perforin. These results suggest that while Bcl-2 is capable of inhibiting the apoptotic pathway utilized by perforin and granzyme B, other granule components can bypass this block. We conclude that CTL harbor potent killing mechanism(s) in addition to those provided by CD95 ligand or perforin and granzyme B that cannot be overcome by Bcl-2.     

Isolation of cDNA encoding full-length rat (Rattus norvegicus) poly (ADP-ribose) polymerase

Poly(ADP-ribose) polymerase (EC 2.4.2.30) is a nuclear enzyme which binds to DNA breaks and then catalyzes the covalent modification of acceptor proteins with poly(ADP-ribose). Poly(ADP-ribose) polymerase activity contributes to the recovery of proliferating cells from DNA damage and to the maintenance of genomic stability, which may be mediated by effects on chromatin structure, DNA base-excision repair and cell cycle regulation. We established the complete cDNA sequence of rat poly(ADP-ribose) polymerase by RT-PCR and direct sequencing of amplification products and compared it with that of other mammalian species. The amino acid sequence homology is strikingly high. The best conserved regions are the known functional modules of poly(ADP-ribose) polymerase.     

Bcl-2 and adenovirus E1B 19 kDA protein prevent E1A-induced processing of CPP32 and cleavage of poly(ADP-ribose) polymerase

The E1A oncoproteins of adenovirus type 5 are potent inducers of apoptotic cell death. To manifest growth promoting and transforming properties, therefore, E1A requires the co-expression of a suppressor of apoptosis. During normal viral infection, this function is provided by the E1B 19 kDa protein. However, the cellular suppressor Bcl-2 can substitute for 19K during infection, and both proteins can effectively cooperate with E1A to facilitate transformation of primary cells in culture. How E1A induces apoptosis and at what point(s) on this pathway Bcl-2 and E1B 19K act are not presently known. Here, we demonstrate that E1A-induced apoptosis is accompanied by specific endo-proteolytic cleavage of poly(ADP-ribose) polymerase (PARP), an event that is linked to the Ced-3/ICE apoptotic pathway in other systems. PARP cleavage was also observed in p53-null cells infected with 19K- virus expressing 13S E1A. In addition to PARP cleavage, expression of E1A caused processing of the zymogen form of CPP32, a Ced-3/ICE protease that cleaves PARP and is required for apoptosis in mammalian cells. These events were prevented when E1A was co-expressed with E1B 19K or BCL-2, which places these suppressors of apoptosis either at or upstream of processing of pro-CPP32.     

Poly(ADP-ribose) polymerase activity is not affected in ataxia telangiectasia cells and knockout mice

Poly(ADP-ribose) polymerase (PARP) is a constitutive factor of the DNA damage surveillance network in dividing cells. Based on its capacity to bind to DNA strand breaks, PARP plays a regulatory role in their resolution in vivo. ATM belongs to a large family of proteins involved in cell cycle progression and checkpoints in response to DNA damage. Both proteins may act as sensors of DNA damage to induce multiple signalling pathways leading to activation of cell cycle checkpoints and DNA repair. To determine a possible relationship between PARP and ATM, we examined the PARP response in an ATM-null background. We demonstrated that ATM deficiency does not affect PARP activity in human cell lines or Atm-deficient mouse tissues, nor does it alter PARP activity induced by oxidative damage or gamma-irradiation. Our results support a model in which PARP and ATM could be involved in distinct pathways, both effectors transducing the damage signal to cell cycle regulators.     

Protease activation and cleavage of poly(ADP-ribose) polymerase: an integral part of apoptosis in response to photodynamic treatment

BACKGROUND & AIMS: We tested the hypothesis that short-chain organic acids in the colon derived from dietary pectin, wheat bran, and oat bran are protective against the development of colon cancer because they induce transforming growth factor (TGF)-beta1, which in turn inhibits cell growth by inducing cyclin-directed kinase (cdk) inhibitors. METHODS: U4 human colon carcinoma cells differentiate into water- and salt-transporting columnar enterocytes and therefore model normal colonocytes. The composition and kinase activity of cdk/cyclin complexes were determined by immunoprecipitation and Western blotting studies in U4 cells treated in vitro with short-chain fatty acid (SCFA) mixtures that mimic the digestion products of wheat bran, oat bran, pectin, and cellulose (as control), which is largely unfermentable. RESULTS: Induction of the cdk inhibitors p21cip1 and p27kip1 by fiber-mimicking SCFA mixtures occurs much more rapidly and is many-fold greater than their induction by TGF-beta1. The SCFA mixtures most effective in causing growth inhibition and cdk inhibitor production mimicked those from wheat bran > oat bran > pectin. CONCLUSIONS: cdk inhibitor induction by SCFA mixtures is not mediated by TGF-beta1. The SCFA mixture mimicking digested wheat bran fiber was the most effective of all mixtures tested in inhibiting cell growth through induction of cdk inhibitors.     

Protease activation during nitric oxide-induced apoptosis: comparison between poly(ADP-ribose) polymerase and U1-70kDa cleavage

Nitric oxide (NO) promotes apoptotic cell death in the mouse macrophage cell line RAW 264.7 and in the human promyelocytic leukaemia cell line U937, which exemplifies p53-dependent and p53-independent executive death pathways. Here, we followed the cleavage of two caspase substrates during NO-intoxication, assaying poly(ADP-ribose) polymerase and U1-70kDa small ribonucleoprotein (U1-70kDa) degradation. By using pharmacological inhibitors, we found that Z-aspartyl-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB; 100 microM), a caspase-like protease inhibitor, completely blocked S-nitrosoglutathione (GSNO)-induced apoptosis in both RAW 264.7 and U937 cells (IC50 = 50 microM for RAW 264.7 macrophages vs. IC50 = 33 microM for U937 cells). Notably, a characterized caspase-3 (Ac-DEVD-CHO) inhibitor left NO-induced DNA fragmentation and the appearance of an apoptotic morphology unaltered, although completely blocking caspase-3 activity. However, Z-Asp-CH2-DCB suppressed protease-mediated U1-70kDa cleavage and DNA fragmentation in parallel. In contrast, poly(ADP-ribose) polymerase cleavage in U937 cells was only delayed by Z-Asp-CH2-DCB, while poly(ADP-ribose) polymerase digestion in RAW 264.7 macrophages proceeded unaltered. We further compared U1-70kDa and poly(ADP-ribose) polymerase cleavage in stably Bcl-2 transfected RAW 264.7 macrophages. Rbcl2-2, a Bcl-2 overexpressing clone, suppressed DNA fragmentation and U1-70kDa digestion in response to GSNO, although allowing delayed but complete poly(ADP-ribose) polymerase degradation. Conclusively, poly(ADP-ribose) polymerase cleavage not causatively coincided with the appearance of other apoptotic parameters. Our results suggest that NO-induced apoptosis demands a Z-Asp-CH2-DCB inhibitable caspase activity, most likely distinct from caspase-3 and caspase-1. NO-mediated executive apoptotic signaling results in U1-70kDa and poly(ADP-ribose) polymerase cleavage. Whereas U1-70kDa digestion closely correlates to the occurrence of apoptotic parameters such as DNA fragmentation or an apoptotic morphology, poly(ADP-ribose) polymerase-breakdown does not.     

Cleavage of poly(ADP-ribose) polymerase: a sensitive parameter to study cell death

Proteases play a crucial role in apoptosis or programmed cell death. The aim of this review is to highlight the purpose for which these proteases are activated, i.e., to specifically cleave a select subset of cellular proteins at an appropriate time during cell death. Poly(ADP-ribose) polymerase (PARP), a nuclear protein implicated in DNA repair, is one of the earliest proteins targeted for a specific cleavage to the signature 89-kDa fragment during apoptosis. Characterization of the apoptotic cleavage of PARP and other target proteins helped in understanding the role of cysteine aspartic acid specific proteases (caspases) in the apoptotic process. We have recently identified that in some models of cell death, the cleavage pattern for PARP is different from production of the signature 89-kDa fragment. Necrotic death of HL-60 cells and apoptotic death of Jurkat cells mediated by granzyme B and perforin were accompanied by distinct additional fragments, suggesting cleavage of PARP at other sites by caspases or other death proteases. This review summarizes how detection and characterization of PARP cleavage could serve as a sensitive parameter for identification of different types of cell death and as a marker for activation of different death proteases. The putative biological functions for early cleavage of PARP in apoptosis are also discussed.