Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

3T3-L1 preadipocytes have been shown to exhibit a transient increase in poly(ADP-ribose) polymerase (PARP) protein and activity, as well as an association of PARP with DNA polymerase alpha, within 12-24 h of exposure to inducers of differentiation, whereas 3T3-L1 cells expressing PARP antisense RNA showed no increase in PARP and are unable to complete the round of DNA replication required for differentiation into adipocytes. The role of PARP in differentiation-linked DNA replication has now been further clarified at both the cellular and enzymological levels. Flow cytometric analysis revealed that control 3T3-L1 cells progressed through one round of DNA replication prior to the onset of terminal differentiation, whereas cells expressing PARP antisense RNA were blocked at the G0/G1 phase of the cell cycle. Confocal microscope image analysis of control S phase cells demonstrated that PARP was localized within distinct intranuclear granular foci associated with DNA replication centers. On the basis of these results, purified replicative complexes from other cell types that had been characterized for their ability to catalyze viral DNA replication in vitro were analyzed for the presence of PARP. PARP exclusively copurified through a series of centrifugation and chromatography steps with core proteins of an 18-21S multiprotein replication complex (MRC) from human HeLa cells, as well as with the corresponding mouse MRC from FM3A cells. The MRC were shown to contain DNA polymerases alpha and delta, DNA primase, DNA helicase, DNA ligase, and topoisomerases I and II, as well as accessory proteins such as PCNA, RF-C, and RP-A. Finally, immunoblot analysis of MRCs from both cell types with monoclonal antibodies to poly (ADP-ribose) revealed the presence of approximately 15 poly(ADP-ribosyl)ated proteins, some of which were further confirmed to be DNA polymerase alpha, DNA topoisomerase I, and PCNA by immunoprecipitation experiments. These results suggest that PARP may play a regulatory role within the replicative apparatus as a molecular nick sensor controlling the progression of the replication fork or modulates component replicative enzymes or factors in the complex by directly associating with them or by catalyzing their poly(ADP-ribosyl)ation.
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PMID:The expression of poly(ADP-ribose) polymerase during differentiation-linked DNA replication reveals that it is a component of the multiprotein DNA replication complex. 879 42

Ara-C has been shown to induce apoptosis of human acute myelogenous leukemia HL-60 cells. The DNA repair enzyme poly(ADP-ribose) polymerase (PARP) is known to be degraded during apoptosis. PARP as a substrate is cleaved by the Yama protease, encoded by the CPP32beta/Yama gene. Yama belongs to the interleukin 1beta converting enzyme/ced-3 family of cysteine proteases that are activated as a cascade, producing proteolytic cleavage of specific substrates that results in the morphological and biochemical features of apoptosis. In the present studies, we determined the effect of high intracellular levels of the antiapoptosis Bcl-2 or Bcl-xL protein on Yama protease activation and PARP degradation during Ara-C-induced apoptosis. For this, we utilized HL-60/Bcl-2, HL-60/Bcl-xL, or control HL-60/neo cells, which were created by transfection of the cDNA of the bcl-2, bcl-xL, or the neomycin-resistant genes, respectively. As compared to HL-60/neo, HL-60/Bcl-2 and HL-60/Bcl-xL cells have 5-fold greater expression of Bcl-2 and Bcl-xL, respectively. However, these cell lines have similar levels of p32Yama and PARP. Treatment with 10 or 100 microM Ara-C for 4 h produced DNA fragmentation and morphological features of apoptosis in HL-60/neo cells. This was associated with the cleavage and activation of p32Yama and PARP degradation but not with the induction of Yama mRNA. In contrast, in HL-60/Bcl-2 and HL-60/ Bcl-xL cells, Ara-C-induced p32Yama activation by its cleavage, PARP degradation and apoptosis were significantly inhibited. High Bcl-2 and Bcl-xL levels in these cells also inhibited Yama protease activity, PARP degradation, and apoptosis due to clinically relevant concentrations of etoposide and mitoxantrone. These results suggest that the activation of the Yama protease and PARP degradation are involved in Ara-C-, etoposide-, or mitoxantrone-induced apoptosis. In addition, they suggest that Bcl-2 and Bcl-xL antagonize drug-induced apoptosis by a mechanism that interferes in the activity of a key cysteine protease that is involved in the execution of apoptosis.
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PMID:Overexpression of Bcl-2 or Bcl-xL inhibits Ara-C-induced CPP32/Yama protease activity and apoptosis of human acute myelogenous leukemia HL-60 cells. 884 Sep 93

Intracellularly, the anticancer drug taxol induces tubulin polymerization and mitotic arrest, followed by apoptosis. The DNA repair enzyme poly(ADP-ribose) polymerase (PARP) and lamins are known to be degraded during apoptosis. PARP is a substrate for the Yama protease, which is encoded by the CPP32 beta/ Yama gene, whereas lamins are degraded by the Yama and lamin proteases. In the present studies, we determined the effects of enforced overexpression of the antiapoptosis Bcl-xL protein on taxol-mediated microtubule and cell cycle perturbations, as well as on taxol-induced apoptosis and associated Yama protease activity in human myeloid leukemia HL-60 cells. Our data demonstrate that high Bcl-xL levels do not affect the microtubular bundling or mitotic arrest due to taxol but significantly inhibit the morphological, flow cytometric, and DNA fragmentation features associated with taxol-induced apoptosis. This resulted in a significant improvement in the survival of taxol-treated cells that possess high Bcl-xL levels. In the control HL-60 cells, following taxol treatment, whereas the mRNA of Yama was not induced, taxol-induced apoptosis was associated with Yama activation and PARP as well as lamin B1 degradation. These features were blocked by coculture of these cells with the cysteine protease inhibitor YVAD-cmk as well as in cells with overexpression of Bcl-xL. These results suggest that Bcl-xL antagonizes taxol-induced apoptosis by a mechanism that interferes with the activation of a key protease involved in the execution of apoptosis.
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PMID:Bcl-xL overexpression inhibits taxol-induced Yama protease activity and apoptosis. 885 5

The activity of some nuclear enzymes associated with DNA repair was examined following aflatoxin B1 administration in rats maintained on different levels of dietary copper. Induction of poly(ADP-ribose) polymerase, DNA polymerase beta and DNA ligase was found to be significantly higher in copper-deficient rats. Copper supplementation, even at marginal doses, was able to bring down the induction to the level observed in normal rats. The results emphasize the protective role of copper against the DNA damaging effects of aflatoxin B1.
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PMID:Modulation by dietary copper of aflatoxin B1-induced activity of DNA repair enzymes poly (ADP-ribose) polymerase, DNA polymerase beta and DNA ligase. 889 34

Interleukin-1beta-converting enzyme (ICE)/Ced-3 proteases play a critical role in apoptosis. One well characterized substrate of these proteases is the DNA repair enzyme poly(ADP-ribose) polymerase. We report here that alpha-fodrin, an abundant membrane-associated cytoskeletal protein, is cleaved rapidly and specifically during Fas- and tumor necrosis factor-induced apoptosis; this cleavage is mediated by an ICE/Ced-3 protease distinct from the poly(ADP-ribose) polymerase protease. Studies in cells treated with these apoptotic stimuli reveal that both fodrin and poly(ADP-ribose) polymerase proteolysis are inhibited by acetyl-Tyr-Val-Ala-Asp chloromethyl ketone and CrmA, specific inhibitors of ICE/Ced-3 proteases. However, fodrin proteolysis can be distinguished from poly(ADP-ribose) polymerase proteolysis by its relative insensitivity to acetyl-Asp-Glu-Val-Asp aldehyde (DEVD-CHO), a selective inhibitor of a subset of ICE/Ced-3 proteases that includes CPP32. DEVD-CHO protects cells from Fas-induced apoptosis but does not prevent fodrin proteolysis, indicating that cleavage of this protein can be uncoupled from apoptotic cell death. Moreover, purified fodrin is cleaved in vitro by CPP32 (but not by ICE) into fragments of the same size observed in vivo during apoptosis. These findings suggest that fodrin proteolysis in vivo may reflect the activity of multiple ICE/Ced-3 proteases whose partial sensitivity to DEVD-CHO reflects a limited contribution from CPP32, or an ICE/Ced-3 protease less sensitive than CPP32 to DEVD-CHO inhibition.
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PMID:Specific cleavage of alpha-fodrin during Fas- and tumor necrosis factor-induced apoptosis is mediated by an interleukin-1beta-converting enzyme/Ced-3 protease distinct from the poly(ADP-ribose) polymerase protease. 894 Jan 32

The aim of this study was to determine whether stable differences in apoptosis sensitivity were selected for in nonmetastatic and metastatic variants of the LNCaP human prostate carcinoma line that had been isolated from tumors grown orthotopically in the prostate glands and regional lymph nodes of nude mice. The nonmetastatic LNCaP-Pro5 cells were significantly more sensitive to thapsigargin-induced apoptosis than were the metastatic LNCaP-LN3 cells, as measured by viability, DNA fragmentation, and interleukin 1beta-converting enzyme family-mediated cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase. Apoptosis resistance in the metastatic cells was associated with higher levels of expression of the cell death suppressor BCL-2 and lower levels of the death promoters BAX and BAK than were detected in the nonmetastatic LNCaP-Pro5 cells, whereas levels of two other BCL-2 family members (BCL-X(L) and BAD) were indistinguishable. Our data support the hypothesis that apoptosis resistance contributes to prostate cancer metastasis and that elevated expression of BCL-2 is involved.
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PMID:Apoptosis resistance increases with metastatic potential in cells of the human LNCaP prostate carcinoma line. 897 Nov 61

Recent evidence suggests that members of the interleukin-1-beta-converting enzyme (ICE)/Ced-3 family are key mediators of mammalian apoptosis. The known members of the ICE/Ced-3 cysteine protease family are synthesized as proenzymes and require proteolytic processing to produce active, heterodimeric enzymes. The baculovirus protein P35 has recently been shown to inhibit several members of the ICE/Ced-3 cysteine protease family. The importance of ICE/Ced-3 cysteine proteases in programmed cell death prompted us to investigate the role of the apoptotic mediator, CPP32, in the glucocorticoid-mediated cell death pathway. Glucocorticoids induce growth inhibition and apoptosis in sensitive leukemic cell lines, immature thymocytes, and eosinophils. In this report, we demonstrate the enzymatic cleavage of proCPP32 to its active subunits in cells undergoing glucocorticoid-induced apoptotic cell death. Concurrently, in apoptotic cells, PARP, a 116-kilodalton (kDa) human poly(ADP-ribose) polymerase, is proteolytically cleaved to its signature 85-kDa fragment. The proteolytic processing of PARP (the nuclear DNA repair enzyme known to be cleaved in association with apoptosis) is catalyzed by members of the ICE/Ced-3 family. Importantly, stable transfection of the antiapoptotic baculovirus P35 inhibits glucocorticoid-induced apoptotic cell death, proteolytic processing of proCPP32, and cleavage of the 116kDa PARP. We conclude that activation of CPP32 is a critical event in glucocorticoid-induced apoptosis and that this pathway is inhibited at or upstream of CPP32 by baculovirus P35. These data demonstrate that PARP cleavage occurs during glucocorticoid-induced apoptotic cell death and show that this proteolytic process is blocked by the expression of baculovirus P35, supporting a role for activation of the ICE/Ced-3-like cysteine protease during glucocorticoid-induced apoptosis.
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PMID:Baculovirus P35 inhibits the glucocorticoid-mediated pathway of cell death. 898 38

Interleukin-1beta-converting enzyme (ICE) is a novel cysteine protease responsible for the cleavage of pre-interleukin-1beta (pre-IL-1beta) to the mature cytokine and a member of a family of related proteases (the caspases) that includes the Caenorhabditis elegans cell death gene product, CED-3. In addition to their sequence homology, these cysteine proteases display an unusual substrate specificity for peptidyl sequences with a P1 aspartate residue. We have examined the kinetics of processing pre-IL-1beta to the mature form by ICE and three of its homologs, TX, CPP-32, and CMH-1. Of the ICE homologs, only TX processes pre-IL-1beta, albeit with a catalytic efficiency 250-fold less than ICE itself. We also investigated the ability of these four proteases to process poly(ADP-ribose) polymerase, a DNA repair enzyme that is cleaved within minutes of the onset of apoptosis. Every caspase examined cleaves PARP, with catalytic efficiencies ranging from 2.3 x 10(6) M-1 s-1 for CPP32 to 1.0 x 10(3) M-1 s-1 for TX. In addition, we report kinetic constants for several reversible inhibitors and irreversible inactivators, which have been used to implicate one or more caspases in the apoptotic proteolysis cascade. Ac-Asp-Glu-Val-Asp aldehyde (DEVD-CHO) is a potent inhibitor of CPP-32 with a Ki value of 0.5 nM, but is also potent as inhibitor of CMH-1 (Ki = 35 nM) and ICE (Ki = 15 nM). The x-ray crystal structure of DEVD-CHO complexed to ICE presented here reveals electrostatic interactions not present in the Ac-YVAD-CHO co-complex structure (Wilson, K. P., Black, J.-A. F., Thomson, J. A., Kim, E. E., Griffith, J. P., Navia, M. A., Murcko, M. A., Chambers, S. P., Aldape, R. A., Raybuck, S. A., and Livingston, D. J. (1994) Nature 370, 270-275), accounting for the surprising potency of this inhibitor against ICE.
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PMID:Substrate and inhibitor specificity of interleukin-1 beta-converting enzyme and related caspases. 905 18

High levels of expression of the DNA repair enzyme O6-alkylguanine DNA-alkyltransferase (OGAT) (EC 2.1.1.63) account for tumor cell resistance to methylating agents. Previous studies suggested that methylating triazenes might have a potential role for the treatment of acute leukemias with low levels of OGAT. In the current study, we transduced the human OGAT cDNA in OGAT-deficient leukemia cell clones. OGAT-transduced cells were more resistant than their OGAT-deficient counterparts to apoptosis triggered by the methylating triazene temozolomide (TZM), as indicated by the results of flow cytometry, terminal deoxynucleotidyl transferase assay, and analysis of DNA fragmentation. Depletion of OGAT activity by O6-benzylguanine increased leukemia cell sensitivity to TZM-mediated apoptosis. Moreover, combined treatment of cells with TZM and benzamide, an inhibitor of the poly(ADP-ribose) polymerase (EC 2.4.2.30), increased the apoptosis induced by the methylating agent. These results demonstrate for the first time that methyl adducts at the O6 position of guanine, which are specifically removed by OGAT, are the principal DNA lesions responsible for the induction of apoptosis on treatment of leukemic cells with the methylating triazene TZM. This study also supports the possible use of TZM for the treatment of acute leukemias and suggests new strategies to increase the susceptibility of tumor cells to methylating triazenes in the clinic.
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PMID:Inhibition of O6-alkylguanine DNA-alkyltransferase or poly(ADP-ribose) polymerase increases susceptibility of leukemic cells to apoptosis induced by temozolomide. 927 47

The DNA repair enzyme O6-alkylguanine DNA-alkyltransferase (OGAT) and a deficient mismatch repair system play a critical role in the resistance to chemotherapeutic agents that generate adducts at the O6-position of guanine. However, DNA adducts different from O6-methylguanine might be also involved in cytotoxicity induced by methylating agents. Because the loss of p53 function is generally associated with tumor cell resistance to anticancer chemotherapy, we have investigated whether wild-type p53 might affect chemosensitivity of leukemia cells endowed with high OGAT levels to the methylating agent temozolomide (TZM). The effect of poly(ADP-ribose) polymerase (PADPRP) inhibition, which potentiates the cytotoxic effects of N7-methylguanine and N3-methylguanine, was also assessed in OGAT-proficient cells, either susceptible or tolerant to O6-methylguanine. OGAT-proficient and p53 null HL60 cells were transfected with the human p53 cDNA (p53+ cells). Treatment with TZM concentrations not toxic for the cells transduced with the control vector (p53-cells), induced apoptosis in p53+ cells. These cells were characterized by a lower level of bcl-2 protein than p53- cells, whereas bax and OGAT expression was comparable in both lines. Inhibition of PADPRP potentiated the cytotoxic and apoptotic effects of TZM in either p53- or p53+ HL60 cells. Furthermore, PADPRP inhibitors potentiated apoptosis induced by TZM in Jurkat cells, which possess a mutated p53 gene and are tolerant to O6-methylguanine adducts. The analysis of cell cycle indicated that the drug combination of TZM and PADPRP inhibitors provoked G1 arrest only in p53+ cells. Conversely, G1 arrest was not observed in p53+ cells exposed to TZM alone. It is possible to speculate that PADPRP inhibitors might affect the repair of DNA adducts that are processed differently from O6 methylguanine and induce a different pattern of cell cycle distribution. In conclusion, the results show that p53 increases apoptosis by TZM in OGAT-proficient cells and suggest the potential role of PADPRP inhibitors in enhancing TZM activity against leukemias independently of DNA repair systems.
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PMID:Role of wild-type p53 on the antineoplastic activity of temozolomide alone or combined with inhibitors of poly(ADP-ribose) polymerase. 958 Jun 40


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