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Human fibroblasts undergo oxidative stress‐induced apoptosis without internucleosomal DNA fragmentation
Author(s) -
Formichi P.,
Radi E.,
Battisti C.,
Tarquini E.,
Leonini A.,
Di Stefano A.,
Federico A.
Publication year - 2006
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.20662
Subject(s) - tunel assay , dna fragmentation , apoptotic dna fragmentation , apoptosis , microbiology and biotechnology , propidium iodide , programmed cell death , fragmentation (computing) , biology , in situ nick end labeling , agarose gel electrophoresis , dna damage , terminal deoxynucleotidyl transferase , dna , biochemistry , ecology
Abstract In order to evaluate the reliability of fibroblasts as a cell model for studying apoptosis, we tested the response of normal human fibroblasts to the oxidative stress inducers H 2 O 2 and 2‐deoxy‐ D ‐ribose (dRib). Our results showed that fibroblasts treated with dRib and H 2 O 2 are induced to undergo apoptosis as demonstrated by reduction in total cell number, chromatin condensation, phosphatidylserine (PS) exposure, activation of caspase‐3 and 7, changes in mitochondrial membrane potential and increase in the number of terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling (TUNEL)‐positive nuclei. However we only found a slight increase in the percentage of cells in the sub‐G1 region evaluated by flow cytometry, and we did not observe DNA fragmentation by agarose gel electrophoresis. Early in apoptosis, DNA cleavage generates high molecular weight (HMW) fragments which can be detected by TUNEL assay; successively followed by a pronounced DNA brake down into low molecular weight (LMW) fragments, detected as a “DNA ladder” by conventional agarose gel electrophoresis and as an hypodiploid peak by propidium iodide (PI) flow cytometry assay. Our results thus suggest that only HMW fragmentation occurs in fibroblasts exposed to dRib or H 2 O 2 and the lack of internucleosomal DNA fragmentation may depend on the peculiar characteristics of human fibroblasts themselves, irrespective of the apoptotic stimulus used. The existence of distinct events leading to cell death in different cell types makes it necessary to use a combination of strategies and techniques to evaluate the occurrence of apoptosis. J. Cell. Physiol. 208: 289–297, 2006. © 2006 Wiley‐Liss, Inc.