Overexpression of deoxyribonuclease I (DNase I) transfected into COS-cells: its distribution during apoptotic cell death.
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.
Polzar
B
B
Institut für Zytobiologie und Zytopathologie, Philipps-Universität, Marburg, Germany.
Peitsch
M C
MC
Loos
R
R
Tschopp
J
J
Mannherz
H G
HG
D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology* / Q000648:ultrastructure; D002843:Chromatin / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000032:analysis; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D003850:Deoxyribonuclease I / Q000032:analysis* / Q000235:genetics* / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic; D006056:Golgi Apparatus / Q000737:chemistry / Q000648:ultrastructure; D010957:Plasmids; D014162:Transfection
D002843:Chromatin; D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D003850:Deoxyribonuclease I