In Vitro Models for Biochemical Investigations on Programmed Cell Death

1991 ◽  
Vol 19 (1) ◽  
pp. 71-76
Author(s):  
Mariarosa Perotti ◽  
Filippo Taddei ◽  
Francesca Mirabelli ◽  
Giorgio Bellomo
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Cold Shock ◽  
In Vitro Models ◽  
Mammary Adenocarcinoma ◽  
Extracellular Medium ◽  
Kinase C ◽  
Intracellular Ca ◽  
Necrosis Factor

Isolated rat thymocytes treated with methylprednisolone (MPS), cultured human synovial (McCoy's) cells exposed to cold shock and human mammary adenocarcinoma (BT-20) cells treated with Tumour Necrosis Factor (TNF), all showed DNA fragmentation and the morphological and biochemical features of cell death characteristic of a process known as apoptosis. DNA fragmentation in MPS-treated thymocytes and in cold shock-exposed McCoy's cells was preceded by a marked increase in cytosolic free Ca2+ concentration resulting either from a sustained influx of Ca2+ from the extracellular medium or from the release of Ca2+ from intracellular pools. On the other hand, in TNF-treated BT-20 cells, DNA fragmentation was not associated with any early, marked increase in the cytosolic free Ca2+ level. In all three models, however, DNA fragmentation was efficiently prevented by intracellular Ca2+ chelators, calmodulin inhibitors and activators of protein kinase C (PKC). These results suggest that the activation of common mechanisms involving Ca2+ and PKC may be essential for the development of apoptosis.

A Relative Energy Failure Is Associated With Low-Mg2+ But Not With 4-Aminopyridine Induced Seizure-Like Events in Entorhinal Cortex

1999 ◽  
Vol 81 (1) ◽  
pp. 399-403 ◽  
Author(s):  
S. Schuchmann ◽  
K. Buchheim ◽  
H. Meierkord ◽  
U. Heinemann
Keyword(s):  
Entorhinal Cortex ◽  
Focal Epilepsy ◽  
Relative Energy ◽  
In Vitro Models ◽  
Mitochondrial Depolarization ◽  
Citrate Cycle ◽  
Cellular Energy ◽  
Intracellular Ca ◽  
Energy Failure

Schuchmann, S., K. Buchheim, H. Meierkord, and U. Heinemann. A relative energy failure is associated with low-Mg2+ but not with 4-aminopyridine induced seizure-like events in entorhinal cortex. J. Neurophysiol. 81: 399–403, 1999. During seizure-like events (SLEs), intracellular Ca2+ concentration ([Ca2+]i) increases causing depolarization of the mitochondrial membrane and subsequent intramitochondrial accumulation of Ca2+. Mitochondrial depolarization results in an interruption of oxidative phosphorylation and increase in reactive oxygen species. Calcium activates enzymes of the citrate cycle. A characteristic feature of the low-Mg2+–induced SLEs is that they are transformed to a late activity refractory to anticonvulsant drugs, which may be regarded as a model system of difficult to treat status epilepticus. In contrast, 4-aminopyridine (4-AP)–induced activity rarely evolves to such late activity. The autofluorescence of NAD(P)H was used to monitor changes in cellular energy metabolism in the entorhinal cortex in two in vitro models of focal epilepsy. During repetitive 4-AP–induced SLEs there was a short decrease followed by a long-lasting overshoot of the NAD(P)H signal. This sequence remained unaltered during recurring SLEs. In contrast, during recurrent low-Mg2+–induced SLEs, the brief initial NADH signal reduction was unchanged but the following overshoot of NADH displayed a continuous decrease. This indicates a relative energy failure, which may contribute to the transformation to late activity in the low-Mg2+ model.

scholarly journals DR5 Knockout Mice Are Compromised in Radiation-Induced Apoptosis

2005 ◽  
Vol 25 (5) ◽  
pp. 2000-2013 ◽  
Author(s):  
Niklas Finnberg ◽  
Joshua J. Gruber ◽  
Peiwen Fei ◽  
Dorothea Rudolph ◽  
Anka Bric ◽  
...  
Keyword(s):  
Cell Death ◽  
Null Mouse ◽  
Organ Specific ◽  
Radiation Induced ◽  
Induced Apoptosis ◽  
The Brain ◽  
Necrosis Factor

ABSTRACT DR5 (also called TRAIL receptor 2 and KILLER) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (also called TRAIL and Apo2 ligand). DR5 is a transcriptional target of p53, and its overexpression induces cell death in vitro. However, the in vivo biology of DR5 has remained largely unexplored. To better understand the role of DR5 in development and in adult tissues, we have created a knockout mouse lacking DR5. This mouse is viable and develops normally with the exception of having an enlarged thymus. We show that DR5 is not expressed in developing embryos but is present in the decidua and chorion early in development. DR5-null mouse embryo fibroblasts expressing E1A are resistant to treatment with TRAIL, suggesting that DR5 may be the primary proapoptotic receptor for TRAIL in the mouse. When exposed to ionizing radiation, DR5-null tissues exhibit reduced amounts of apoptosis compared to wild-type thymus, spleen, Peyer's patches, and the white matter of the brain. In the ileum, colon, and stomach, DR5 deficiency was associated with a subtle phenotype of radiation-induced cell death. These results indicate that DR5 has a limited role during embryogenesis and early stages of development but plays an organ-specific role in the response to DNA-damaging stimuli.

Lens Epithelial Cells Express CD95 and CD95 Ligand Treatment Induces Cell death and DNA Fragmentation in Vitro

2003 ◽  
Vol 13 (3) ◽  
pp. 241-245 ◽  
Author(s):  
A. Hueber ◽  
C.D. Eichholtz ◽  
N. Kociok ◽  
J.M. Esser ◽  
P.J. Esser
Keyword(s):  
Cell Death ◽  
Epithelial Cells ◽  
Dna Fragmentation ◽  
Lens Epithelial Cells ◽  
Cd95 Ligand

Alpha 1-adrenoceptor and purinoceptor agonists modulate Na-H antiport in single cardiac cells

1993 ◽  
Vol 264 (2) ◽  
pp. H310-H319 ◽  
Author(s):  
M. Puceat ◽  
O. Clement-Chomienne ◽  
A. Terzic ◽  
G. Vassort
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cardiac Cells ◽  
Extracellular Medium ◽  
Beta Adrenoceptor ◽  
Kinase C ◽  
Intracellular Ca ◽  
Acid Load ◽  
Ethanesulfonic Acid ◽  
Stimulation Of

We investigated the effects of an alpha 1-adrenoceptor (phenylephrine) and a purinoceptor agonist (ATP), both of which accelerate the phosphoinositide turnover, on the Na-H antiport activity of rat single cardiac cells using the pH-sensitive fluorescent indicator seminaphthorhodafluor-1 (SNARF-1). Both phenylephrine, in the presence of a beta-adrenoceptor blocker, and ATP enhanced the ability of the cell to regulate its intracellular pH (pHi) after an imposed acid load. This effect was observed in HCO3-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and prevented by Na-H antiport inhibitors ethylisopropylamiloride (EIPA) or amiloride. Similar results were obtained when cells were bathed in an acidic extracellular medium. Hence, the alpha 1-adrenoceptor and purinoceptor agonists activate the Na-H antiport even when it is partially inhibited by extracellular protons. To further evaluate the effects of the two neurohormones, the rate of proton efflux was estimated as a function of the magnitude of the imposed acid load. The results indicate that the agonist-induced modulation of the Na-H antiport is caused by an acceleration of its exchange activity and by a shift of its dependence on pHi toward more alkaline pH values. The agonist-mediated stimulation of the antiport was also observed in partially depolarized cells and was not dependent on intracellular Ca. Phorbol 12-myristate 13-acetate was not able to reproduce the effects of the agonists on the Na-H antiport. Conversely, the inhibitors of protein kinase C did not prevent the activation of the antiport by the neurohormones. Thus our data suggest that neither a Ca-calmodulin-dependent kinase nor protein kinase C is responsible for the alpha 1-adrenoceptor- and purinoceptor-mediated stimulation of the antiport.

Increased cell death in rat blastocysts exposed to maternal diabetes in utero and to high glucose or tumor necrosis factor-alpha in vitro

Development ◽  
1997 ◽  
Vol 124 (23) ◽  
pp. 4827-4836 ◽  
Author(s):  
S. Pampfer ◽  
I. Vanderheyden ◽  
J.E. McCracken ◽  
J. Vesela ◽  
R. De Hertogh
Keyword(s):  
Cell Death ◽  
Tumor Necrosis ◽  
Maternal Diabetes ◽  
Diabetic Rats ◽  
Tnf Alpha ◽  
Nuclear Fragmentation ◽  
Necrosis Factor Alpha ◽  
Factor Alpha ◽  
Necrosis Factor

The morphogenetic function of the transient phase of cell death that occurs during blastocyst maturation is not known but it is thought that its regulation results from a delicate balance between survival and lethal signals in the uterine milieu. In this paper, we show that blastocysts from diabetic rats have a higher incidence of dead cells than control embryos. Differential lineage staining indicated that increased nuclear fragmentation occurred mainly in the inner cell mass. In addition, terminal transferase-mediated dUTP nick end labeling (TUNEL) demonstrated an increase in the incidence of non-fragmented DNA-damaged nuclei in these blastocysts. Analysis of the expression of clusterin, a gene associated with apoptosis, by quantitative reverse transcription-polymerase chain reaction detected an increase in the steady-state level of its transcripts in blastocysts from diabetic rats. In situ hybridization revealed that about half the cells identified as expressing clusterin mRNA exhibited signs of nuclear fragmentation. In vitro experiments demonstrated that high D-glucose increased nuclear fragmentation, TUNEL labeling and clusterin transcription. Tumor necrosis factor-alpha (TNF-alpha), a cytokine whose synthesis is up-regulated in the diabetic uterus, did not induce nuclear fragmentation nor clusterin expression but increased the incidence of TUNEL-positive nuclei. The data suggest that excessive cell death in the blastocyst, most probably resulting from the overstimulation of a basal suicidal program by such inducers as glucose and TNF-alpha, may be a contributing factor of the early embryopathy associated with maternal diabetes.

scholarly journals Development of an Ischemic Tolerance Model in a PC12 Cell Line

2005 ◽  
Vol 25 (2) ◽  
pp. 154-162 ◽  
Author(s):  
Joëlle A Hillion ◽  
Kenzo Takahashi ◽  
Dragan Maric ◽  
Christl Ruetzler ◽  
Jeffery L Barker ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Pc12 Cell ◽  
Annexin V ◽  
Glucose Deprivation ◽  
In Vitro Models ◽  
Ischemic Tolerance ◽  
Cellular Mechanisms ◽  
Pc12 Cell Line

Although ischemic tolerance has been described in a variety of primary cell culture systems, no similar in vitro models have been reported with any cell line. A model of ischemic preconditioning in the rat pheochromocytoma PC12 cell line is described here. When compared to nonpreconditioned cells, preexposure of PC12 cells to 6 hours of oxygen and glucose deprivation (OGD) significantly increased cell viability after 15 hours of OGD 24 hours later. Flow cytometry analysis of cells labeled with specific markers for apoptosis, Annexin V, and Hoechst 33342, and of DNA content, revealed that apoptosis is involved in OGD-induced PC12 cell death and that preconditioning of the cells mainly counteracts the effect of apoptosis. Immunocytochemistry of caspase-3, a central executioner in the apoptotic process, further confirmed the activation of apoptotic pathways in OGD-induced PC12 cell death. This model may be useful to investigate the cellular mechanisms involved in neuronal transient tolerance following ischemia.

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