Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells.

Abstract

Human diploid fibroblast cells lose replicative potential after a certain number of population doublings. We use this experimental system to investigate the role of oxidative damage in cellular aging. Treating cells with H202 at <300 JAM did not affect the viability of the majority of cells when judged by morphology, trypan blue exclusion, and protein synthesis. However, the treatment caused a dosedependent inhibition of DNA synthesis. After a 2-hr treatment with 200 FM H202, the cells failed to respond to a stimulus of serum, platelet-derived growth factor, basic fibroblast growth factor, or epidermal growth factor by synthesizing DNA, and the loss of response could not be recovered by 4 days. Subcultivation showed that, as in senescent cells, division of the treated cells was inhibited. The life-time cumulative growth curve showed that the loss of replication due to H202 treatment was cumulative and irreversible. The H202 treatment decrased the number of the population doublings in the rest of the life span by 35.3 ± 10.3%. Enzymatic assays indicated that, like the cells in their senescent state, the treated cells were less able to activate ornithine decarboxylase and thymidine kinase. Furthermore, subcultivation after the H202 treatment showed that the cells developed the morphology of senescent cells. In conclusion, sublethal treatment of H202 "stuned" F65 cells and caused the cells to enter a state resembling senescence.One of the manifestations of aging is the accumulation of damage at both cellular and organismal levels. This damage is initiated by endogenous and exogenous toxicants. Oxidants appear to be the major source of damage and are produced during normal aerobic metabolism and inflammatory reactions (1, 2). High-dose exposure to oxidants may cause cytotoxicity and neoplastic transformation. We investigate here the role of oxidants in aging with human diploid fibroblast (HDF) cells as an experimental system. HDF cells lose replicative capacity in culture after a specific number ofpopulation doublings (PDs) and have been widely used as an in vitro model of aging (3)(4)(5)(6). Accompanying the loss of replicative potential, senescence is marked by several changes, including decreased activity of cellcycle-related enzymes (9, 10) and decreased efficiency of protein synthesis and degradation (11)(12)(13)(14). Nuclear and chromosomal aberrations are present at a higher frequency (15,16). The content of 5-methylcytosine in DNA and the length of telomeres are decreased (17)(18)(19) Amersham or 20 Ci/mmol from NEN) at 0.5 gCi per well or a 14C-labeled mixture of amino acids (54.4 mCi/mol atom carbon from NEN) at 0.2 uCi per well. The labeling medium was washed out and the cells were fixed in the well with 10% (wt/vol) ice-cold trichloroacetic acid. After two additional washes with 10o trichloroacetic acid, one wash with 70% ethanol, and one wash with phosphate-buffered saline (PBS), the precipitates were dissolved in 0.1 M KOH. Trichloroacetic acid-insoluble radioactivity was determined by liquid s...

UVB-induced premature senescence of human diploid skin fibroblasts

“…The ratios of incorporation between the cells exposed to repeated stresses or not are very similar to those observed in cells exposed to a single subcytotoxic stress at 450 M H 2 O 2 [16] or to five repeated subcytotoxic stresses at 30 M t-BHP [11]. Using this criteria too, we can suggest that the UVB stressed HDFs behave like presenescent cells.…”

AMPK activation protects cells from oxidative stress‐induced senescence via autophagic flux restoration and intracellular NAD + elevation

“…H 2 O 2 treatment‐induced fibroblast senescence has been widely used as a model of SIPS (Chen & Amos, 1994; Toussaint et al ., 2000). With a modified procedure, we obtained H 2 O 2 ‐induced senescence in NIH3T3 cells with good homogeneity.…”

“…Cellular senescence can occur spontaneously in vivo and in vitro , also can be induced in vitro when cells are exposed to oxidative stress, such as hydrogen peroxide (H 2 O 2 ) (Chen & Amos, 1994; Toussaint et al ., 2000). This type of senescence is commonly referred as to oxidative stress‐induced senescence (SIPS).…”

Pancreatic beta cell senescence contributes to the pathogenesis of type 2 diabetes in high-fat diet-induced diabetic mice

“…Correlations between cellular senescence and AUC insulin (c) (r= −0.852, p<0.01), insulinogenic index (d) (r=−0.857, p<0.05), and phospho-p38-positive beta cells (e) (r=0.843, p<0.05) of the high-fat diet group after 12 months by enhancing the activity of MAPK, which consists of at least three enzymes, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. For example, in rat kidney cells the ageing process parallels increases in ROS that strongly enhance the activities of ERK, JNK, and p38 MAPK [21]. Isawa et al reported that p38 MAPK is an important causative molecule in both telomere-dependent and telomere-independent cellular senescence, including ROS-induced senescence in both human and mouse fibroblasts [37].…”

“…Mammalian cells, with the exceptions of germ line cells and stem cells, have a limited replication potential in vivo, defined as replicative senescence, which amounts to irreversible growth arrest after a limited number of cell divisions [19]. Cellular senescence is also associated with reactive oxygen species (ROS), the incessant damaging products generated from aerobic metabolism such as oxidative stress [20][21][22][23]. During the pathogenesis of type 2 diabetes, beta cell proliferation increases to compensate for the increased insulin demand caused by insulin resistance, and the generation of ROS is induced by hyperglycaemia [6-8, 12, 24].…”