Overexpression of deoxyribonuclease I (DNase I) transfected into COS-cells: its distribution during apoptotic cell death

Détails

ID Serval
serval:BIB_C1963DD0AA06
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Overexpression of deoxyribonuclease I (DNase I) transfected into COS-cells: its distribution during apoptotic cell death
Périodique
European Journal of Cell Biology
Auteur(s)
Polzar  B., Peitsch  M. C., Loos  R., Tschopp  J., Mannherz  H. G.
ISSN
0171-9335 (Print)
Statut éditorial
Publié
Date de publication
12/1993
Volume
62
Numéro
2
Pages
397-405
Notes
Journal Article
Research Support, Non-U.S. Gov't --- Old month value: Dec
Résumé
COS-cells were transiently transfected with the pSG5 plasmid containing the cDNA of rat parotid deoxyribonuclease I (DNase I) either in right or inverse orientation. Expression of DNase I in transfected cells was only observed when the plasmid contained the cDNA in the right orientation. Expression of DNase I was monitored by measuring the DNase I specific DNA-degrading activity present in the conditioned cell culture medium and in cell homogenates. The expressed DNase I activity could be inhibited by monospecific polyclonal antibodies and by G-actin. Immunofluorescence indicated that approximately 20% of the COS-cells transfected with the DNase I-cDNA in right orientation expressed DNase I. These transfected cells contained large amounts of DNase I, which was found to be localized within the rough endoplasmic reticulum, the Golgi-complex and finally concentrated in a perinuclear location. Occasionally cells were observed which contained the DNase I in small apparently secretory transport vesicles. Transfected cells with perinuclear concentration of DNase I exhibited progressive nuclear destruction, i.e., pyknosis and cytoplasmic shrinkage. Solely the DNA extracted from isolated nuclei of cells transfected with the DNase I-cDNA in correct orientation revealed an internucleosomal DNA-degradation (ladder formation) typical for apoptosis after incubation in the presence of CaCl2 and MgCl2. Only the conditioned medium of COS-cells transfected with the right-oriented DNase I-cDNA contained the nucleolytic activity able to internucleosomally degrade the chromatin of substrate nuclei. Thus, these results indicate that overexpression of DNase I alone is sufficient to induce the morphological and biochemical changes observed during apoptosis.
Mots-clé
Animals *Apoptosis Cell Line Cell Nucleus/*enzymology/ultrastructure Chromatin/metabolism Culture Media, Conditioned/analysis DNA, Complementary/analysis/genetics Deoxyribonuclease I/*analysis/*genetics/physiology Endoplasmic Reticulum/chemistry/ultrastructure Fluorescent Antibody Technique Gene Expression Regulation, Enzymologic Golgi Apparatus/chemistry/ultrastructure Plasmids Transfection
Pubmed
Web of science
Création de la notice
24/01/2008 16:19
Dernière modification de la notice
03/03/2018 21:08
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