A recombinant cytotoxic chimera based on mammalian deoxyribonuclease-I

Linardou, Helena; Epenetos, Agamemnon A.; Deonarain, Mahendra P.

doi:10.1002/(sici)1097-0215(20000515)86:4<561::aid-ijc19>3.0.co;2-5

Cited by 16 publications

(8 citation statements)

References 32 publications

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“…However, this correction was not applied in the report by Savchenko et al (52). Another reason we chose the low dose is based on the concern that DNase I is a proapoptotic agent (2, 31, 48, 66) and has been used in cancer treatment (35). In the setting of myocardial ischemia, apoptosis was thought to be an important contributor to cardiomyocyte death (60).…”

Section: H505mentioning

confidence: 99%

Neutrophil extracellular traps in ischemia-reperfusion injury-induced myocardial no-reflow: therapeutic potential of DNase-based reperfusion strategy

Zhou

et al. 2015

American Journal of Physiology-Heart and Circulatory Physiology

201

135

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Emerging evidence suggests a potential role of neutrophil extracellular traps (NETs) in linking sterile inflammation and thrombosis. We hypothesized that NETs would be induced during myocardial ischemia-reperfusion (I/R), and NET-mediated microthrombosis may contribute to myocardial "no-reflow". Male Wistar rats were randomly divided into I/R control, DNase (DNase I, 20 μg/rat), recombinant tissue-type plasminogen activator (rt-PA, 420 μg/rat), DNase + rt-PA, and sham control groups after 45-min myocardial ischemia. In situ NET formation, the anatomic "no re-flow" area, and infarct size were evaluated immediately after 3 h of reperfusion. Long-term left ventricular (LV) functional and histological analyses were performed 45 days after operation. Compared with the I/R controls, the DNase + rt-PA group exhibited reduced NET density [8.38 ± 1.98 vs. 26.86 ± 3.07 (per 200 × field), P < 0.001] and "no-flow" area (15.22 ± 0.06 vs. 34.6 ± 0.05%, P < 0.05) in the ischemic region, as well as reduced infarct size (38.39 ± 0.05 vs. 71.00 ± 0.03%, P < 0.001). Additionally, compared with the I/R controls, DNase + rt-PA treatment significantly ameliorated I/R injury-induced LV remodeling (LV ejection fraction: 64.22 ± 3.37 vs. 33.81 ± 2.98%, P < 0.05; LV maximal slope of the LV systolic pressure increment: 3,785 ± 216 vs. 2,596 ± 299 mmHg/s, P < 0.05). The beneficial effect was not observed in rats treated with DNase I or rt-PA alone. Our study provides evidence for the existence of NETs in I/R-challenged myocardium and confirms the long-term benefit of a novel DNase-based reperfusion strategy (DNase I + rt-PA), which might be a promising option for the treatment of myocardial I/R injury and coronary no-reflow.

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Section: H505mentioning

confidence: 99%

Neutrophil extracellular traps in ischemia-reperfusion injury-induced myocardial no-reflow: therapeutic potential of DNase-based reperfusion strategy

Zhou

et al. 2015

American Journal of Physiology-Heart and Circulatory Physiology

201

135

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“…The eluted proteins were neutralised with 0.2 vol of 1 M phosphate buffer, pH 7.4 and dialysed exhaustively against 150 mM citrate phosphate buffer, pH 5 or 6, or against PBS. Linamarase expressed from clone pYXCas5-HA-HIS was purified on an immobilised copper-(II)-chelating sepharose column by immobilised metal affinity chromatography using a method similar to that described in Linardou et al 17 …”

Section: Isolation and Purification Of Recombinant Proteinsmentioning

confidence: 99%

“…Detection was with 1 g/ml anti-haemagglutinin (Boehringer Mannheim, Indianapolis, IN) or anti-penta-HIS antibody (Qiagen, Chatsworth, CA) followed by anti-rat horseradish peroxidase-conjugated IgG or anti-mouse horseradish peroxidase-conjugated IgG, respectively, as described in Linardou et al 17 Binding of tested proteins on live cells was demonstrated by fluorescence-activated cell sorting (FACS) analyses. For each analysis, 10 6 cells were washed and blocked with PBS/2% FCS.…”

Section: Antigen-binding Activity Of the Fusion Proteinmentioning

confidence: 99%

Antibody‐guided enzyme therapy of cancer producing cyanide results in necrosis of targeted cells

Kousparou¹,

Epenetos²,

Deonarain³

2002

Intl Journal of Cancer

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A number of enzyme/prodrug activation approaches for the treatment of cancer have been reported to date with varying success. We describe progress in the development of a system based on a ␤-glucosidase enzyme in combination with a naturally occurring "prodrug," the sugar linamarin, which releases the cytotoxin cyanide. A recombinant fusion protein, composed of an scFv (MFE-23) reactive against carcinoembryonic antigen (CEA) and a plant-derived ␤-glucosidase (linamarase), was produced and its cytotoxic potential was investigated. The fusion protein was expressed in a supersecretory mutant strain of Saccharomyces cerevisiae and purified by affinity chromatography. Extensive functional in vitro characterisation of the fusion protein showed that it retained antigen binding activity but that its catalytic activity was impaired, a problem not related to its fusion with the scFv. Nevertheless, we demonstrated complete tumour cell killing at doses of prodrug that are completely nontoxic to nontargeted cells. Preliminary in vivo characterisation showed that extensive glycosylation of the fusion protein caused its rapid clearance through the hepatic route. Aggregational properties also led to poor pharmacokinetics. Furthermore, we present some data analysing the mode of cell death resulting from exposure to this system. Enzymic catalysis of the substrate generates cyanide, a metabolic poison that asphyxiates cells and leads them to a necrotic-like cell death. This system has been called antibody-guided enzyme nitrile therapy (AGENT).

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“…DNase γ, an actin‐resistant DNase I‐like endonuclease, may also have clinical benefits for cystic fibrosis patients (Shiokawa and Tanuma 2001). A chimeric molecule comprising an scFv (immunoreactive against the human placental alkaline phosphatase) and bpDNase was also designed and investigated for its cytotoxic potential and possible use as immunotoxin in tumor‐targeting strategies in cancer therapy (Linardou et al 2000). In the present study, the mutant with a newly engineered disulfide [brDNase(F192C/A217C)] was proved to be thermostable and resistant against trypsin inactivation.…”

Section: Discussionmentioning

confidence: 99%

Biological functions of the disulfides in bovine pancreatic deoxyribonuclease

et al. 2004

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We characterized the biochemical functions of the small nonessential (C101-C104) and the large essential (C173-C209) disulfides in bovine pancreatic (bp) DNase using alanine mutants [brDNase(C101A)] and [brDNase(C173A) and brDNase(C209A)], respectively. We also characterized the effects of an additional third disulfide [brDNase(F192C/A217C)]. Without the Ca 2+ protection, bpDNase and brDNase(C101A) were readily inactivated by trypsin, whereas brDNase(F192C/A217C) remained active. With Ca 2+ , all forms of DNase, except for brDNase(C101A), were protected against trypsin. All forms of DNase, after being dissolved in 6 M guanidine-HCl, were fully reactivated by diluting into a Ca 2+ -containing buffer. However, when diluted into a Ca 2+ -free buffer, bpDNase and brDNase(C101A) remained inactive, but 60% of the bpDNase activity was restored with brDNase(F192C/A217C). When heated, bpDNase was inactivated at a transition temperature of 65°C, brDNase(C101A) at 60°C, and brDNase(F192C/A217C) at 73°C, indicating that the small disulfide, albeit not essential for activity, is important for the structural integrity, and that the introduction of a third disulfide can further stabilize the enzyme. When pellets of brDNase(C173A) and brDNase(C209A) in inclusion bodies were dissolved in 6 M guanidine-HCl and then diluted into a Ca 2+ -containing buffer, 10%-18% of the bpDNase activity was restored, suggesting that the "essential" disulfide is not absolutely crucial for enzymatic catalysis. Owing to the structure-based sequence alignment revealing homology between the "nonessential" disulfide of bpDNase and the active-site motif of thioredoxin, we measured 39% of the thioredoxin-like activity for bpDNase based on the rate of insulin precipitation (⌬A650nm/min). Thus, the disulfides in bpDNase not only play the role of stabilizing the protein molecule but also may engage in biological functions such as the disulfide/dithiol exchange reaction.

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A recombinant cytotoxic chimera based on mammalian deoxyribonuclease-I

Cited by 16 publications

References 32 publications

Neutrophil extracellular traps in ischemia-reperfusion injury-induced myocardial no-reflow: therapeutic potential of DNase-based reperfusion strategy

Neutrophil extracellular traps in ischemia-reperfusion injury-induced myocardial no-reflow: therapeutic potential of DNase-based reperfusion strategy

Antibody‐guided enzyme therapy of cancer producing cyanide results in necrosis of targeted cells

Biological functions of the disulfides in bovine pancreatic deoxyribonuclease

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