Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Aug 15;47(4):410-8.
doi: 10.1016/j.freeradbiomed.2009.05.003. Epub 2009 May 8.

Early Onset Senescence Occurs When Fibroblasts Lack the Glutamate-Cysteine Ligase Modifier Subunit

Affiliations
Free PMC article

Early Onset Senescence Occurs When Fibroblasts Lack the Glutamate-Cysteine Ligase Modifier Subunit

Ying Chen et al. Free Radic Biol Med. .
Free PMC article

Abstract

Cellular senescence is the irreversible entry of cells into growth arrest. Senescence of primary cells in culture has long been used as an in vitro model for aging. Glutamate-cysteine ligase (GCL) controls the synthetic rate of the important cellular antioxidant glutathione (GSH). The catalytic subunit of GCL, GCLC, is catalytically active and essential for life. By contrast the modifier subunit of GCL, GCLM, is dispensable in mice. Although it is recognized that GCLM increases the rate of GSH synthesis, its physiological role is unclear. Herein, we show that loss of Gclm leads to premature senescence of primary murine fibroblasts as characterized by: (a) diminished growth rate, (b) cell morphology consistent with senescence, (c) increases in senescence-associated beta-galactosidase activity, and (d) cell cycle arrest at the G(1)/S and G(2)/M boundaries. These changes are accompanied by increased intracellular ROS, accumulation of DNA damage, and induction of p53 and p21 proteins. We also found that N-acetylcysteine increases intracellular GSH and prevents premature senescence in Gclm(-/-) cells. These results suggest that the control of GCLM, which in turn controls aspects of the cellular redox environment via GSH, is important in determining the replicative capacity of the cell.

Figures

Fig. 1
Fig. 1
Decreased proliferative span and growth rate in Gclm(−/−) MFFs. The proliferative capacity is expressed as population-doubling (PD) numbers, derived from the equation as described in “Materials and Methods”. Gclm(−/−) cells revealed growth retardation starting at P5 and a smaller maximal PD (MPD). Data are reported as means ± S.E. for six to eight individual wells of MFF cultures for each group, and three independent experiments. *p <0.05, comparing Gclm(+/+) and Gclm(−/−) cells at the same passage number.
Fig. 2
Fig. 2
Premature senescence morphology and increased SA-β-gal activity in Gclm(−/−) MFFs. (A) General morphology of the MFF cells. Starting at passage 5 (P5), Gclm(−/−) cells became enlarged and flattened, with a decreased nucleus-to-cytoplasm ratio. (400x) (B) Representative images of SA-β-gal staining in Gclm(+/+) and Gclm(−/−) cells at P7. (400x) (C) Quantification of the SA-β-gal-staining. The number of positive (blue stained) cells was counted in 200 cells and SA-β-gal activity was expressed as percent of positive cells in total cells. *: p <0.05, comparing Gclm(+/+) and Gclm(−/−) cells at the same passage number.
Fig. 3
Fig. 3
Growth arrest at the G1/S and G2/M boundaries in senescent Gclm(−/−) MFFs. Upper left: Population-doubling (PD) time (in hours) was determined from the growth rate assay as described in “Materials and Methods”. Lower left and at right: Cell population (%) in each cell-cycle phase was determined by flow-cytometry. Data are reported as means ± S.E. of three or four individual wells of cell cultures for each group. *p <0.05, comparing Gclm(+/+) and Gclm(−/−) cells at the same passage number. ^p <0.05, compared with P1 cells of the same genotype.
Fig. 4
Fig. 4
Intracellular GSH levels and response to H2O2 in Gclm(+/+) versus Gclm(−/−) MFFs. (A) Intracellular GSH levels at P1 through P9 were determined spectrophotofluorometrically using o-phthalaldehyde. (B) Cell viability, subsequent to H2O2 treatment for 8 h, was determined by the MTT assay and expressed as percent of untreated cells (100% viable). Data are reported as means ± S.E. for three or four individual wells of MFF cultures for each group.
Fig. 5
Fig. 5
ROS formation and DNA damage in Gclm(+/+) versus Gclm(−/−) MFFs. (A) Intracellular ROS levels were determined using H2O2-activated CM-H2DCFDA as the fluorescent probe by flow cytometry. Cells in the absence of probe (unstained) were used to generate negative baseline. Fluorescent intensity in P1 Gclm(+/+) wild-type cells was set as the control; ratio to this control level is expressed as the relative ROS level. (B) DNA strand breaks were determined by the alkaline comet assay. Comet was scored from 0 to 4, based on the length of the comet tail. The extent of DNA damage was expressed as the total comet score in 150–200 cells, as described in “Materials and Methods”. P1 Gclm(+/+) cells, treated with 100 μM H2O2 for 2 h, was used as the positive control. Data are reported as means ± S.E. of three or four individual wells of MFF cultures for each group. *p <0.05, comparing Gclm(+/+) and Gclm(−/−) cells at the same passage number, ^p <0.05, compared with P1 cells of the same genotype.
Fig. 6
Fig. 6
Induction of p53 and p21 in senescent Gclm(−/−) MFFs. (A) The mRNA levels of p53 and p21 were quantified by Q-PCR. The mRNA level for each gene in P1 Gclm(+/+) cells was set as the control (= 1.0). Relative mRNA levels are expressed as fold of the control after normalization with β-Actin. Data are reported as means ± S.E. of 3–4 individual cell cultures for each group. *p <0.05, comparing Gclm(+/+) and Gclm(−/−) cells at the same passage number; ^p <0.05, compared with P1 cells of the same genotype. (B) Semi-quantification of p53 and p21 proteins in Gclm(+/+) versus Gclm(−/−) cells. Western immunoblot analysis was carried out on p53, p21 and β-actin in nuclear extracts (30 μg) from cells. Inductions of p53 and p21 were observed in senescent Gclm(−/−) cells (P7).
Fig. 7
Fig. 7
Prevention of premature senescence in Gclm(−/−) MFFs by NAC supplementation. Freshly made NAC (final concentration 5 mM) was added to the standard culture medium starting at P1. (A) Intracellular GSH levels in NAC-treated Gclm(−/−) cells approach those seen in Gclm(+/+) cells. (B) Intracellular ROS levels were lowered by NAC treatment in Gclm(−/−) cells, but remained higher than those in Gclm(+/+) cells—with or without NAC treatment. (C) DNA strand breaks remained at control levels in NAC-treated Gclm(−/−) cells, compared with NAC-treated Gclm(+/+) cells. (D) Similar growth profiles were observed in NAC-treated Gclm(+/+) and NAC-treated Gclm(−/−) cells. (E) Pre-senescent cell morphology and negligible SA-β-gal-positive staining are the same in P9 NAC-treated cells of both genotypes. Data are reported as means ± S.E. of three or four individual wells of MFF cultures for each group. *p <0.05, comparing untreated and NAC-treated MFFs of the same genotype at the same passage number.

Similar articles

See all similar articles

Cited by 11 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback