Quercetin-Induced PC12 Cell Death Accompanied by Caspase-Mediated DNA Fragmentation

Serum-free PC12 cell cultures have been used to study the mechanisms of neuronal death after neurotrophic factor deprivation. We previously reported that PC12 cells undergo "apoptotic" internucleosomal DNA cleavage after withdrawal of trophic support. Here, we have used a sensitive method to detect PC12 cell DNA fragmentation within three hrs of serum removal and have exploited this assay to examine several aspects regarding the mechanisms of neuronal survival/death. Major advantages of this assay are that it permits acute experiments to be performed well before other manifest signs of cell death and under conditions that cannot be applied chronically. We find that this apopotic DNA fragmentation is distinct from the random DNA degradation that occurs during necrotic death. Major observations include the following: (a) There is a good correlation between the ability of trophic substances to promote PC12 cell survival and to inhibit early DNA fragmentation. (b) Phorbol ester, an activator of PKC, acutely suppresses DNA fragmentation, but does not promote long-term survival or inhibition of endonuclease activity when applied chronically due to its downregulation of PKC. (c) Cells undergoing apoptosis within 3 h of serum withdrawal have a "commitment point" of only 1.0-1.5 h beyond which they can no longer be rescued by NGF. (d) Aurin, a non-carboxylic analog of the endonuclease inhibitor ATA, also inhibits DNA fragmentation and promotes short-term survival of PC12 cells. (e) Macromolecular synthesis is not required for DNA fragmentation or for NGF to prevent this event. (f) Extracellular Ca2+ is not required for internucleosomal DNA cleavage caused by serum withdrawal or for suppression of this by NGF. (g) DNA fragmentation can also be detected in cultures of rat sympathetic neurons as early as 10 h after removal of NGF. As in PC12 cell cultures, this precedes morphological signs of cell death.

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Etoposide-induced PC12 cell death: apoptotic morphology without oligonucleosomal DNA fragmentation or dependency upon de novo protein synthesis

Molecular Brain Research ◽

10.1016/s0169-328x(97)00105-8 ◽

1997 ◽

Vol 48 (2) ◽

pp. 382-388 ◽

Cited By ~ 14

Author(s):

Josep Saura ◽

Geraldine MacGibbon ◽

Mike Dragunow

Keyword(s):

Cell Death ◽

Protein Synthesis ◽

Dna Fragmentation ◽

Pc12 Cell ◽

De Novo ◽

Apoptotic Morphology ◽

De Novo Protein Synthesis

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TUNEL Assay: A Powerful Tool for Kidney Injury Evaluation

International Journal of Molecular Sciences ◽

10.3390/ijms22010412 ◽

2021 ◽

Vol 22 (1) ◽

pp. 412

Author(s):

Christopher L. Moore ◽

Alena V. Savenka ◽

Alexei G. Basnakian

Keyword(s):

Cell Death ◽

Dna Fragmentation ◽

Cell Injury ◽

Kidney Injury ◽

Cell Types ◽

Tunel Assay ◽

Terminal Deoxynucleotidyl Transferase ◽

Hypoxic Injury ◽

Additional Information ◽

Tissue Compartments

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay is a long-established assay used to detect cell death-associated DNA fragmentation (3’-OH DNA termini) by endonucleases. Because these enzymes are particularly active in the kidney, TUNEL is widely used to identify and quantify DNA fragmentation and cell death in cultured kidney cells and animal and human kidneys resulting from toxic or hypoxic injury. The early characterization of TUNEL as an apoptotic assay has led to numerous misinterpretations of the mechanisms of kidney cell injury. Nevertheless, TUNEL is becoming increasingly popular for kidney injury assessment because it can be used universally in cultured and tissue cells and for all mechanisms of cell death. Furthermore, it is sensitive, accurate, quantitative, easily linked to particular cells or tissue compartments, and can be combined with immunohistochemistry to allow reliable identification of cell types or likely mechanisms of cell death. Traditionally, TUNEL analysis has been limited to the presence or absence of a TUNEL signal. However, additional information on the mechanism of cell death can be obtained from the analysis of TUNEL patterns.

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