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Comparative characterization of rat deoxyribonuclease 1 (Dnase1) and murine deoxyribonuclease 1-like 3 (Dnase1l3).

ABSTRACT: Deoxyribonuclease 1 (DNASE1, DNase I) and deoxyribonuclease 1-like 3 (DNASE1L3, DNase gamma, DNase Y, LS-DNase) are members of a DNASE1 protein family that is defined by similar biochemical properties such as Ca2+/Mg2+-dependency and an optimal pH of about 7.0 as well as by a high similarity in their nucleic acid and amino acid sequences. In the present study we describe the recombinant expression of rat Dnase1 and murine Dnase1l3 as fusion proteins tagged by their C-terminus to green fluorescent protein in NIH-3T3 fibroblasts and bovine lens epithelial cells. Both enzymes were translocated into the rough endoplasmic reticulum, transported along the entire secretory pathway and finally secreted into the cell culture medium. No nuclear occurrence of the nucleases was detectable. However, deletion of the N-terminal signal peptide of both nucleases resulted in a cytoplasmic and nuclear distribution of both fusion proteins. Dnase1 preferentially hydrolysed 'naked' plasmid DNA, whereas Dnase1l3 cleaved nuclear DNA with high activity. Dnase1l3 was able to cleave chromatin in an internucleosomal manner without proteolytic help. By contrast, Dnase1 was only able to achieve this cleavage pattern in the presence of proteases that hydrolysed chromatin-bound proteins. Detailed analysis of murine sera derived from Dnase1 knockout mice revealed that serum contains, besides the major serum nuclease Dnase1, an additional Dnase1l3-like nucleolytic activity, which, in co-operation with Dnase1, might help to suppress anti-DNA autoimmunity by degrading nuclear chromatin released from dying cells.

SUBMITTER: Napirei M

PROVIDER: S-EPMC1175112 | biostudies-literature | 2005 Jul

REPOSITORIES: biostudies-literature

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Comparative characterization of rat deoxyribonuclease 1 (Dnase1) and murine deoxyribonuclease 1-like 3 (Dnase1l3).

Napirei Markus M Wulf Swantje S Eulitz Dirk D Mannherz Hans Georg HG Kloeckl Thomas T

The Biochemical journal 20050701 Pt 2

Deoxyribonuclease 1 (DNASE1, DNase I) and deoxyribonuclease 1-like 3 (DNASE1L3, DNase gamma, DNase Y, LS-DNase) are members of a DNASE1 protein family that is defined by similar biochemical properties such as Ca2+/Mg2+-dependency and an optimal pH of about 7.0 as well as by a high similarity in their nucleic acid and amino acid sequences. In the present study we describe the recombinant expression of rat Dnase1 and murine Dnase1l3 as fusion proteins tagged by their C-terminus to green fluorescen ...[more]

PMID: 15796714

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Project description:IntroductionOvarian cancer is the most deadly among all gynecological cancers. Patients undergoing systemic therapies of advanced ovarian cancers suffer from horrendous side effects. Cancer survivors and their offspring suffer from iatrogenic consequences of systemic therapies: genetic mutations. The ultimate goal of our work is development of therapies, which selectively and completely eliminate cancer cells, but do not harm healthy cells. An important consideration for attaining this goal is the fact that ovarian cancer cells over-express EGFR or its mutants, what becomes the factor discriminating them from healthy cells - a potential facilitator of personalized therapy.Specific aimThe specific aim of this project was threefold: (1) to bioengineer suicide genes' carrying vectors guided by synthetic antibodies for EGFRvIII and EGFR; (2) to genetically engineer DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, and DFFB controlled by the EGFR promoter; (3) to selectively eradicate ovarian cancer cells by intranuclear targeting of the transgenically expressed recombinant DNases.MethodsSynthetic antibodies for EGFR and EGFRvIII were selected from the human library and used to bioengineer biotag-guided transgenes' vectors. Coding sequences for the human DNASE1, DNASE1L3, DNASE2, DFFB controlled by the EGFR promoter were amplified from the human cDNA and genetically engineered into the plasmid constructs also coding for the fusions with NLS and GFP. The vectors carrying transgenes for the DNases were delivered in vitro into human ovarian cancer cells from ascites and cultures.ResultsSynthetic antibody guided vectors delivered the transgenes for the recombinant DNases efficiently into the ovarian cancer cells. Transgenic expression and nuclear targeting of the DNases in those cells resulted in destruction of their genomes and led to their death, as validated by labeling with the molecular death tags. In healthy cells, which did not over-express EGFR, no changes were recorded.ConclusionTargeted expression of the recombinant DNASE1, DNASE1L3, DNASE2, DFFB in the ovarian cancers in vitro resulted in their complete eradication, but had no effects upon the healthy cells. This novel therapeutic strategy has a potential for streamlining it into in vivo trials, as personalized, targeted therapy of ovarian and other cancers.

Project description:The worst possible complication of using stem cells for regenerative therapy is iatrogenic cancerogenesis. The ultimate goal of our work is to develop a self-triggering feedback mechanism aimed at causing death of all stem cells, which resist directed differentiation, keep proliferating, and can grow into tumors. The specific aim was threefold: (1) to genetically engineer the DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter; (2) to bioengineer anti-SSEA-4 antibody guided vectors delivering transgenes to human undifferentiated and proliferating pluripotent stem cells; (3) to cause death of proliferating and directed differentiation resisting stem cells by transgenic expression of the human recombinant the DNases (hrDNases). The DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter were genetically engineered. The vectors targeting specifically SSEA-4 expressing stem cells were bioengineered. The healthy volunteers' bone marrow mononuclear cells (BMMCs) were induced into human, autologous, pluripotent stem cells with non-integrating plasmids. Directed differentiation of the induced stem cells into endothelial cells was accomplished with EGF and BMP. The anti-SSEA 4 antibodies' guided DNA vectors delivered the transgenes for the human recombinant DNases' into proliferating stem cells. Differentiation of the pluripotent induced stem cells into the endothelial cells was verified by highlighting formation of tight and adherens junctions through transgenic expression of recombinant fluorescent fusion proteins: VE cadherin, claudin, zona occludens 1, and catenin. Proliferation of the stem cells was determined through highlighting transgenic expression of recombinant fluorescent proteins controlled by POLA promoter, while also reporting expression of the transgenes for the hrDNases. Expression of the transgenes for the DNases resulted in complete collapse of the chromatin architecture and degradation of the proliferating cells' genomic DNA. The proliferating stem cells, but not the differentiating ones, were effectively induced to die. Herein, we describe attaining the proof-of-concept for the strategy, whereby transgenic expression of the genetically engineered human recombinant DNases in proliferating and directed differentiation resisting stem cells leads to their death. This novel strategy reduces the risk of iatrogenic neoplasms in stem cell therapy.

Dataset Information

Comparative characterization of rat deoxyribonuclease 1 (Dnase1) and murine deoxyribonuclease 1-like 3 (Dnase1l3).

Publications

Comparative characterization of rat deoxyribonuclease 1 (Dnase1) and murine deoxyribonuclease 1-like 3 (Dnase1l3).

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