Human fibroblasts undergo oxidative stress-induced apoptosis without internucleosomal DNA fragmentation.
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.
Formichi
P
P
Department of Neurological and Behavioural Sciences, University of Siena, Siena, Italy.
Radi
E
E
Battisti
C
C
Tarquini
E
E
Leonini
A
A
Di Stefano
A
A
Federico
A
A
D017209:Apoptosis / Q000187:drug effects*; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D053938:DNA Fragmentation; D003855:Deoxyribose / Q000494:pharmacology; D005347:Fibroblasts / Q000473:pathology* / Q000502:physiology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D009707:Nucleosomes; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D013997:Time Factors
D009707:Nucleosomes; D016877:Oxidants; D003855:Deoxyribose; D006861:Hydrogen Peroxide