Human fibroblasts undergo oxidative stress‐induced apoptosis without internucleosomal DNA fragmentation

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.

Hydrogen peroxide enhanced Ca2+-activated BK currents and promoted cell injury in human dermal fibroblasts

“…In addition, it has been previously reported that H 2 O 2 stimulates BK channels through the NO/ sGC/cGMP pathway in cultured human endothelial cells and HEK293 cells that express BK channels (Dong et al, 2008;Ma et al, 2010). Therefore, these observations indicate that the same ion channel could be Although it has been accepted that apoptosis is characterised by distinct biochemical and morphological hallmarks, data from Formichi et al revealed that human dermal fibroblasts apoptosis induced by oxidising agents may occur in the absence of one of the characteristic features of apoptosis, namely internucleosomal DNA fragmentation (Formichi et al, 2006). In our experiment, we also did not observe H 2 O 2 -induced DNA fragmentation by agarose gel electrophoresis (data not show), the results are similar to those reported by Formichi et al These different cell behaviours may depend on the specific characteristics of human fibroblasts and may be related to the apoptotic stimulus used (Formichi et al, 2006).…”

“…Therefore, these observations indicate that the same ion channel could be Although it has been accepted that apoptosis is characterised by distinct biochemical and morphological hallmarks, data from Formichi et al revealed that human dermal fibroblasts apoptosis induced by oxidising agents may occur in the absence of one of the characteristic features of apoptosis, namely internucleosomal DNA fragmentation (Formichi et al, 2006). In our experiment, we also did not observe H 2 O 2 -induced DNA fragmentation by agarose gel electrophoresis (data not show), the results are similar to those reported by Formichi et al These different cell behaviours may depend on the specific characteristics of human fibroblasts and may be related to the apoptotic stimulus used (Formichi et al, 2006). In this study, we thus detected the effect of H 2 O 2 -enhanced K + current on cell injury in human dermal fibroblasts using a combination of PI and TUNEL assay staining, which indicated the cell death and apoptosis damage, respectively.…”

Influence of chlorine dioxide on cell death and cell cycle of human gingival fibroblasts

“…Excessive apoptosis has been related to sepsis, ischemic heart disease, stroke, and neurodegenerative diseases, while functional deficiencies in the activation of apoptosis may facilitate the development of cancer and autoimmune diseases. 34 We examined the effects of disinfectants on cell death using an annexin V-FITC/PI assay at close to the LD 50 concentration (0.05, 0.1, and 0.5 mM). The dose-independent effect showed that H 2 O 2 , but not ClO 2 or NaOCl, induced cell death by producing an apoptotic signal at 0.05 and 0.1 mM (Figs.…”

Effects of high-dose major components in oral disinfectants on the cell cycle and apoptosis in primary human gingival fibroblasts in vitro

“…The deregulation of apoptosis is implicated in a variety of diseases. Excessive apoptosis is related to sepsis, ischemic heart disease, stroke, and neurodegenerative diseases, and functional deficiencies in the activation of apoptosis may facilitate the development of certain cancers and autoimmune diseases 32) . During the early stages of apoptosis, a loss of phospholipid asymmetry occurs that results in the translocation of negatively charged phosphatidylserine (PS) from the inner to the outer leaflet of the cell membrane 33) .…”