Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To elucidate the biological functions of poly(ADP-ribose) polymerase (PARP, [EC 2.4.2.30]) in DNA damage responses, genetic and biochemical approaches were undertaken. By disrupting exon 1 of the mouse PARP gene by a homologous recombination, PARP-deficient mouse embryonic stem (ES) cell lines and mice could be produced without demonstrating lethality. PARP-/- ES cells showed complete loss of PARP activity and increased sensitivity to gamma-irradiation and an alkylating agents, indicating a physiological role for PARP in the response to DNA damage. p53, a key molecule in cellular DNA damage response, was found to stimulate PARP activity and became poly(ADP-ribosyl)ated in the presence of damaged DNA. However, PARP-/- ES cells showed p21 and Mdm-2 mRNA induction following gamma-irradiation, indicating that PARP activity is not indispensable for p21 and Mdm-2 mRNA induction in the established p53-cascade. On the other hand, in a reconstituted reaction system, purified PARP from human placenta suppressed the pRB-phosphorylation activity in the presence of NAD and damaged DNA. Human PARP expressed in E. coli showed a similar effect on pRB-phosphorylation activity of cdk2. These findings suggest a direct involvement of PARP in the regulation of cdk activity for cell-cycle arrest.
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PMID:Function of poly(ADP-ribose) polymerase in response to DNA damage: gene-disruption study in mice. 1033 51

Caspases are responsible for the proteolysis of many cytoskeletal proteins in apoptotic cells. It has been demonstrated here that during cisplatin-induced apoptosis of human embryo retinoblasts both E- and P-cadherin were degraded by caspases, giving initially major polypeptide products of apparent molecular weights 48 K and 104 K respectively. This proteolysis occurred over a similar time-scale to the observed degradation of PARP and to the onset of DNA fragmentation but appreciably later than p53 induction and cleavage of Mdm2 and p21. Addition of caspase inhibitors such as Z-VAD-FMK inhibited apoptosis and cadherin degradation. Co-immunoprecipitation studies carried out on viable cells confirmed previously observed complexes between cadherins and alpha and beta catenin and between the catenins themselves. These interactions were sustained in apoptotic cells as long as the protein components remained intact. Using confocal microscopy it has been shown that cytoskeletal changes associated with apoptosis precede degradation of catenins and cadherins by several hours. In particular, after addition of cisplatin relatively rapid (within 3 h) re-localization of adherens junction proteins from the cell periphery to the cytoplasm was observed whereas little cadherin or catenin degradation occurred until 10 h. We conclude that neither caspase-mediated degradation of cytoskeletal components nor disruption of adherens junction protein-protein interactions is required for morphological change.
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PMID:The fate of E- and P-cadherin during the early stages of apoptosis. 1038 31

Although several studies have suggested that inhibition of arterial narrowing by radiation after angioplasty is dependent on both time and dose, little is known regarding the temporal aspects of this effect and the mechanisms by which radiation affects the response of smooth muscle cells to injury. To determine the time course of inhibition of intimal hyperplasia by radiation, 135 rats were given single-fraction external gamma irradiation (1-10 Gy) to one carotid artery at intervals from 5 days before to 5 days after bilateral carotid artery balloon catheter injury, and intimal cross-sectional area was determined from histological sections at 20 days after injury. There was a prominent time- and dose-dependent inhibition of intimal hyperplasia by radiation when it was administered before or after balloon injury, with the greatest effect noted within 24 h before or after injury. To investigate the effect of radiation on smooth muscle cell growth (by cell counting) and proliferation, cell cycle kinetics (by BrdU incorporation), and cell killing (by clonogenic assay), smooth muscle cell cultures derived from rat aortic explants were seeded in equine plasma to induce quiescence, and radiation (2.5-10 Gy) was administered at various intervals before or after synchronous growth stimulation by 10% whole blood serum. A similar time and dose dependence was noted in growth kinetics, BrdU incorporation and cell killing for smooth muscle cells irradiated in vitro; in each case, the effect was most prominent for radiation administered in temporal proximity to stimulation with whole blood serum. By Western blot analysis, cultured smooth muscle cells showed a rapid time-dependent increase in Cdkn1a (formerly known as p21) protein expression, followed by a delayed increase in Tp53 (formerly known as p53) expression after irradiation. Activation of intracellular caspases, manifest by proteolytic poly(ADP-ribose) polymerase (PARP) cleavage, was not detected in smooth muscle cell cultures after irradiation. These observations suggest that radiation limits intimal hyperplasia in vivo by a transient, reversible process. Although apparent cytotoxic injury occurs in vitro, apoptosis of smooth muscle cells is not apparent. Both inhibition of proliferation of smooth muscle cells and cell cycle delay may contribute to inhibition of intimal hyperplasia in vivo by radiation.
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PMID:Inhibition of rat smooth muscle proliferation by radiation after arterial injury: temporal characteristics in vivo and in vitro. 1062 14

Pyrrolidine dithiocarbamate (PDTC) is a synthetic antioxidant molecule, which has been recently proposed as an antitumoral agent on the basis of its capability of inducing apoptosis. We investigated the effect of PDTC on the proliferation and survival of the promyelocitic cell line HL-60. Concentration as low as 10 microM of PDTC induces a significant reduction of the growth rate and the contemporaneous activation of the apoptotic process. Programmed cell death was demonstrated by biochemical analyses, including the activation of procaspase 3 and the cleavage of poly(ADP-ribose) polymerase (PARP). PDTC-dependent apoptosis was associated with an early release of cytochrome c from mitochondria, while the involvement of pathways due to cell death receptors engagement was ruled out by detailed time-course analyses of caspases 3 and 8 activation. Moreover, no up-regulation of p21(CIP1) level, a pivotal cyclin-dependent kinase inhibitor, occurred at PDTC concentration able to induce apoptosis. Finally, in vitro incubation of purified mitochondria with PDTC demonstrated that the molecule is directly able to induce cytochrome c release from the intermembrane space, thus confirming that mitochondria are a primary cellular target of the molecule.
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PMID:Pyrrolidine dithiocarbamate induces apoptosis by a cytochrome c-dependent mechanism. 1067 10

Death receptors of the Tumor Necrosis Factor (TNF) family form membrane-bound self-activating signaling complexes that initiate apoptosis through cleavage of proximal caspases including CASP8 and 10. Here we show that overexpression of the cytoplasmic domain (CD) of the DR4 TRAIL receptor (TNFRSF10A, TRAIL R1) in human breast, lung, and colon cancer cell lines, using an adenovirus vector (Ad-DR4-CD), leads to p53-independent apoptotic cell death involving cleavage of CASP8 and 10 proximally and CASP3, 6, and 7 distally. DR4-CD overexpression also leads to cleavage of poly(ADP-ribose) polymerase (PARP) and the DNA fragmentation factor (DFF45; ICAD). Importantly, normal lung fibroblasts are resistant to DR4-CD overexpression and show no evidence of PARP-, CASP8- or CASP3-cleavage despite similar levels of adenovirus-delivered DR4-CD protein as the cancer cells. These results suggest that DR4 may signal death through known caspases and that further studies are required to evaluate Ad-DR4-CD as a novel anti-cancer agent. Finally, we show that overexpression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (CDKN1A), or its N-terminal 91 amino acids containing cell cycle-inhibitory activity, inhibits DR4-CD-dependent proximal caspase cleavage. The blockage of initiator caspase activation provides a novel insight into how p21 may suppress apoptosis and enhance cell survival.
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PMID:p21(WAF1/CIP1) inhibits initiator caspase cleavage by TRAIL death receptor DR4. 1069 97

The human prostatic carcinoma cell line LNCaP is sensitive to TNF-alpha treatment and expresses wild-type p53. To analyse the possible role of p53 in TNF-alpha-mediated apoptosis, we generated a derivative of LNCaP, LN-56, expressing a dominant-negative element of p53, GSE56. P53 inactivation in LN-56 was associated with an increased resistance to apoptosis induced by TNF-alpha. Surface expression of TNF-alpha receptors was unchanged in LN-56 compared to LNCaP. TNF-alpha treatment resulted in accumulation of p53 in LNCaP and upregulation of p21/WAF1. Activation of caspase-7 and PARP proteolysis were delayed in LN-56 under TNF-alpha treatment. TNF-alpha-induced apoptosis in LNCaP cells was accompanied by caspase-dependent proteolysis of p21/WAF1 and Rb, which was significantly attenuated in LN-56. Cytochrome c release was induced by TNF-alpha treatment in both cell lines, but caspase-9 was not activated. LNCaP and LN-56 were injected s.c. in nude mice and tumors were identified in all LN-56, but not LNCaP, bearing mice indicating that p53 plays an important role in growth control of prostatic neoplasms. Interestingly, accumulation of p53 in TNF-alpha-treated LNCaP cells was decreased in the presence of the caspase inhibitor Z-VAD-FMK, suggesting a new role of activated caspases in acceleration of p53 response. In summary, these results indicate that p53 is involved in TNF-alpha-mediated apoptosis in LNCaP.
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PMID:p53 is involved in tumor necrosis factor-alpha-induced apoptosis in the human prostatic carcinoma cell line LNCaP. 1077 86

Photodynamic therapy (PDT) using the silicon phthalocyanine photosensitizer Pc 4 [HOSiPcOSi(CH3)2(CH2)3N-(CH3)2] is an oxidative stress associated with induction of apoptosis in various cell types. We assessed the effectiveness of Pc 4-PDT on SW480 colon cancer xenografts grown in athymic nude mice. Animals bearing xenografts were treated with 1 mg/kg body weight Pc 4 and 48 h later were irradiated with 150 J/cm2 672-nm light from a diode laser delivered at 150 mW/cm2. Biochemical studies were performed in xenografts resected at various time points up to 26 h after Pc 4-PDT treatment, whereas tumor size was evaluated over a 4-week period in parallel experiments. In the tumors resected for biochemical studies, apoptosis was visualized by activation of caspase-9 and caspase-3 and a gradual increase in the cleavage of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) to a maximum of approximately 60% of the total PARP present at approximately 26 h. At that time all Pc 4-PDT-treated tumors had regressed significantly. Two signaling responses that have previously been shown to be associated with Pc 4-PDT-induced apoptosis in cultured cells, p38 mitogen-activated protein kinase and p21/WAF1/Cip1, were examined. A marked increase in phosphorylation of p38 was observed within 1 h after Pc 4-PDT without changes in levels of the p38 protein. Levels of p21 were not altered in the xenografts in correspondence with the presence of mutant p53 in SW480 cells. Evaluation of tumor size showed that tumor growth resumed after a delay of 9-15 days. Our results suggest that: (a) Pc 4-PDT is effective in the treatment of SW480 human colon cancer xenografts independent of p53 status; (b) PARP cleavage may be mediated by caspase-9 and caspase-3 activation in the Pc 4-PDT-treated tumors; and (c) p38 phosphorylation may be a trigger of apoptosis in response to PDT in vivo in this tumor model.
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PMID:Photodynamic therapy with the phthalocyanine photosensitizer Pc 4 of SW480 human colon cancer xenografts in athymic mice. 1081 28

The mechanism(s) of c-Myc transcription factor-induced apoptosis is still obscure. The activation of c-Myc has been found to lead into the processing/activation of caspases (caspase-3), but the significance of this for the cell demise is debatable. Here we report that several targets of caspases (PKCdelta, MDM2, PARP, replication factor C, 70 kDa U1snRNP, fodrin and lamins) are cleaved during c-Myc-induced apoptosis in Rat-1 MycER cells, indicating an important role for caspases in the apoptotic process. We further found that the ATM (ataxia telangiectasia mutated)--protein is a novel key substrate of caspases. In in vitro assays, purified recombinant ATM protein was found to be cleaved by the effector caspases 3 and 7. The functional significance of the ATM cleavage is supported by the finding that ectopic expression of ATM protected in part against apoptosis. We also show that c-Myc-induced apoptosis involves loss of mitochondrial transmembrane potential, release of cytochrome c from mitochondria into the cytosol and subsequent processing of caspase-9. The cleavage of caspase-9 is, however, minimal and a much later event than the processing/activation of caspase-3, suggesting that it is not the apical caspase. Evidence is provided that there is, nevertheless, an upstream caspase(s) regulating the functions of caspase-3 and mitochondria. Additionally, it was found that p53 becomes upregulated, together with its transcriptional targets MDM2 and p21, upon c-Myc induction, but this occurs also at a later time than the activation of caspase-3.
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PMID:Caspases and mitochondria in c-Myc-induced apoptosis: identification of ATM as a new target of caspases. 1082 87

In over 90% of cervical cancers and cancer-derived cell lines, the p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV). The HPV E6 protein promotes the degradation of p53 and thus inhibits the stabilization and activation of p53 that would normally occur in response to HPV E7 oncogene expression. Restoration of p53 function in these cells by blocking this pathway should promote a selective therapeutic affect. Here we show that treatment with the small molecule nuclear export inhibitor, leptomycin B, and actinomycin D leads to the accumulation of transcriptionally active p53 in the nucleus of HeLa, CaSki, and SiHa cells. Northern blot analyses showed that both actinomycin D and leptomycin B reduced the amount of HPV E6-E7 mRNA whereas combined treatment with the drugs showed almost complete disappearance of the viral mRNA. The combined treatment activated p53-dependant transcription, and increases in both p21(WAF1/CIP1) and Hdm2 mRNA were seen. The combined treatment resulted in apoptotic death in the cells, as evidenced by nuclear fragmentation and PARP-cleavage indicative of caspase 3 activity. These effects were greatly reduced by expressing a dominant negative p53 protein. The present study shows that small molecules can reactivate p53 in cervical carcinoma cells, and this reactivation is associated with an extensive biological response, including the induction of the apoptotic death of the cells.
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PMID:Activation of p53 in cervical carcinoma cells by small molecules. 1090 10

Inhibitors of proteases are currently emerging as a potential anti-cancer modality. Nonselective protease inhibitors are cytotoxic to leukemia and cancer cell lines and we found that this cytotoxicity is correlated with their potency as inhibitors of the proteasome but not as inhibitors of calpain and cathepsin. Highly selective inhibitors of the proteasome were more cytotoxic and fast-acting than less selective inhibitors (PS341>>ALLN>>ALLM). Induction of wt p53 correlated with inhibition of the proteasome and antiproliferative effect in MCF7, a breast cancer cell line, which was resistant to apoptosis caused by proteasome inhibitors. In contrast, inhibitors of the proteasome induced apoptosis in four leukemia cell lines lacking wt p53. The order of sensitivity of leukemia cells was: Jurkat>HL60> or =U937>>K562. The highly selective proteasome inhibitor PS-341 induced cell death with an IC50 as low as 5 nM in apoptosis-prone leukemia cells. Cell death was preceded by p21WAF1/CIP1 accumulation, an alternative marker of proteasome inhibition, and by cleavage of PARP and Rb proteins and nuclear fragmentation. Inhibition of caspases abrogated PARP cleavage and nuclear fragmentation and delayed, but did not completely prevent cell death caused by PS-341. Reintroduction of wt p53 into p53-null PC3 prostate carcinoma cells did not increase their sensitivity to proteasome inhibitors. Likewise, comparison of parental and p21-deficient cells demonstrated that p21WAF1/CIP1 was dispensable for proteasome inhibitor-induced cytotoxicity. We conclude that accumulation of wt p53 and induction of apoptosis are independent markers of proteasome inhibition.
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PMID:Protease inhibitor-induced apoptosis: accumulation of wt p53, p21WAF1/CIP1, and induction of apoptosis are independent markers of proteasome inhibition. 1091 53


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