PMC

Selenoproteins are differentially modulated by selenium levels and replicative senescence. Selenoprotein expression level was evaluated by immunoblotting in extracts from young cultivated for two passages in Dpl, Ctrl, and Sup media in comparison with senescent cells grown in Ctrl conditions and normalized over actin signal. The relative expression is indicated, with the Ctrl condition being set at 1. Data are sorted as down-regulated (A), poorly affected (B), and up-regulated (C) in response to replicative senescence.

Analysis of replicative life span of WI-38 cells as a function of selenium concentration. A, young human embryonic lung fibroblasts WI-38 (CPD36) were cultivated in Dpl, Dpl+Se, Ctrl, and Sup conditions and evaluated for their proliferative capacity using CPD calculation. Shown is an enlargement of the first three passages in the different media. Cells are considered senescent when the CPD reaches a plateau. ○, Dpl; ▵, Dpl+Se; ◆, Ctrl; □, Sup. B, representative pictures of young cells grown for two passages in the different media and of senescent cells.

Analysis of Gpx and TR activities as a function of selenium levels and replicative senescence. Activities of Gpx (A) and TR (B) were measured in protein extracts from young cells (gray bars) cultivated for two passages in Dpl, Ctrl, and Sup media in comparison with senescent cells grown in Ctrl and Sup conditions (black bars). The data are represented as the mean of three independent experiments ± S.D. (error bars). The differences between activities that are significant (*, p < 0.05) are indicated above the respective brackets in -fold change.

Selenoprotein mRNAs levels as a function of selenium levels and replicative senescence. A, evolution of mRNA levels as a function of selenium levels in young cells. B, comparison of mRNA abundance between young and senescent WI-38 cells grown in Ctrl conditions. The steady state levels of mRNAs were measured by RT-qPCR and normalized over Hrpt in total RNA extracts. Data are represented as the mean of three independent experiments ± S.D. (error bars) in logarithmic scale. The differences between activities that are significant (*, p < 0.05) are indicated above the respective brackets in -fold change. C, representation of common mRNA targets between selenium variation and replicative senescence.

The response of UGA/Sec recoding efficiency as a function of selenium levels and replicative senescence depends on the nature of the SECIS element. A, representation of the luciferase reporter assay used for the analysis of UGA recoding efficiency. The open reading frame of firefly luciferase with a UGA/Sec codon at position 258 is linked to different SECIS elements. A control construct of the Luc UGA/SelX was made, where the UGA was replaced by a UGU/cysteine. B, young cells were cultivated for two passages in either Dpl, Ctrl, or Sup medium and transfected with luciferase plasmid. Luciferase activities obtained with UGA construct were normalized relative to the signal measured from LucUGU/Gpx4 transfection. Data from three independent experiments were arbitrarily expressed relative to the activity obtained in young Ctrl extracts (set at 100%) ± S.D. (error bars). C, comparison of the UGA/Sec recoding efficiencies between young and senescent fibroblasts grown in Ctrl medium. D, cellular localization of SBP2 in young and senescent cells.

Senescence-associated characteristics and signaling pathways are modified in young WI-38 cells by the selenium levels. Young cells (gray bars) were cultivated for two passages in Dpl, Ctrl, and Sup medium and evaluated for senescence-associated markers in comparison with senescent cells grown in Ctrl conditions (black bars). A, the percentage of SAHF-positive cells is represented for the different growth conditions. B, the proportion of cells positive for SABG was measured in the different growth media. C, the length of telomeric DNA was quantified by qPCR on genomic DNA and normalized relative to a single copy gene (β-globin). The data are represented as the mean of three independent experiments ± S.D. (error bars). The significant -fold change differences between the different growth conditions and/or proliferative state are indicated above the respective brackets (p < 0.05). D, protein extracts from young WI-38 cells were evaluated for p16, p21, pRb, and p53 expression levels by immunoblotting, relative to actin (Ctrl condition being set at 1). E, these protein extracts were also were analyzed by oxiblot in triplicates. The mean of the total signal from each lane is indicated below the immunoblot. Apparent molecular mass markers are indicated in the right side in kDa.