Young WI-38 Cells Research Articles

Interplay between Selenium Levels and Replicative Senescence in WI-38 Human Fibroblasts: A Proteomic Approach.

Selenoproteins are essential components of antioxidant defense, redox homeostasis, and cell signaling in mammals, where selenium is found in the form of a rare amino acid, selenocysteine. Selenium, which is often limited both in food intake and cell culture media, is a strong regulator of selenoprotein expression and selenoenzyme activity. Aging is a slow, complex, and multifactorial process, resulting in a gradual and irreversible decline of various functions of the body. Several cellular aspects of organismal aging are recapitulated in the replicative senescence of cultured human diploid fibroblasts, such as embryonic lung fibroblast WI-38 cells. We previously reported that the long-term growth of young WI-38 cells with high (supplemented), moderate (control), or low (depleted) concentrations of selenium in the culture medium impacts their replicative lifespan, due to rapid changes in replicative senescence-associated markers and signaling pathways. In order to gain insight into the molecular link between selenium levels and replicative senescence, in the present work, we have applied a quantitative proteomic approach based on 2-Dimensional Differential in-Gel Electrophoresis (2D-DIGE) to the study of young and presenescent cells grown in selenium-supplemented, control, or depleted media. Applying a restrictive cut-off (spot intensity ±50% and a p value < 0.05) to the 2D-DIGE analyses revealed 81 differentially expressed protein spots, from which 123 proteins of interest were identified by mass spectrometry. We compared the changes in protein abundance for three different conditions: (i) spots varying between young and presenescent cells, (ii) spots varying in response to selenium concentration in young cells, and (iii) spots varying in response to selenium concentration in presenescent cells. Interestingly, a 72% overlap between the impact of senescence and selenium was observed in our proteomic results, demonstrating a strong interplay between selenium, selenoproteins, and replicative senescence.

Anti-ageing effects of Sonchus oleraceus L. (pūhā) leaf extracts on H₂O₂-induced cell senescence.

Antioxidants protect against damage from free radicals and are believed to slow the ageing process. Previously, we have reported the high antioxidant activity of 70% methanolic Sonchus oleraceus L. (Asteraceae) leaf extracts. We hypothesize that S. oleraceus extracts protect cells against H2O2-induced senescence by mediating oxidative stress. Premature senescence of young WI-38 cells was induced by application of H2O2. Cells were treated with S. oleraceus extracts before or after H2O2 stress. The senescence- associated β-galactosidase (SA-β-gal) activity was used to indicate cell senescence. S. oleraceus extracts showed higher cellular antioxidant activity than chlorogenic acid in WI-38 cells. S. oleraceus extracts suppressed H2O2 stress-induced premature senescence in a concentration-dependent manner. At 5 and 20 mg/mL, S. oleraceus extracts showed better or equivalent effects of reducing stress-induced premature senescence than the corresponding ascorbic acid treatments. These findings indicate the potential of S. oleraceus extracts to be formulated as an anti-ageing agent.

Phosphorylation of HuR by Chk2 Regulates SIRT1 Expression

The RNA binding protein HuR regulates the stability of many target mRNAs. Here, we report that HuR associated with the 3' untranslated region of the mRNA encoding the longevity and stress-response protein SIRT1, stabilized the SIRT1 mRNA, and increased SIRT1 expression levels. Unexpectedly, oxidative stress triggered the dissociation of the [HuR-SIRT1 mRNA] complex, in turn promoting SIRT1 mRNA decay, reducing SIRT1 abundance, and lowering cell survival. The cell cycle checkpoint kinase Chk2 was activated by H(2)O(2), interacted with HuR, and was predicted to phosphorylate HuR at residues S88, S100, and T118. Mutation of these residues revealed a complex pattern of HuR binding, with S100 appearing to be important for [HuR-SIRT1 mRNA] dissociation after H(2)O(2). Our findings demonstrate that HuR regulates SIRT1 expression, underscore functional links between the two stress-response proteins, and implicate Chk2 in these processes.

Senescent WI-38 Fibroblasts Overexpress LPC-1, a Putative Transmembrane Shock Protein

We have recently reported the isolation of cDNAs for a number of genes that are differentially expressed between nonproliferating early (young) and late (senescent) population doubling level (PDL) WI-38 human, fetal lung-derived, fibroblast-like cells. We now demonstrate that one of these isolates, LPC-1 (Late PDL cDNA-1), derives from an approximately 2.9-kb mRNA species that is expressed at a two- to fivefold higher level in serum-starved, confluent, senescent versus similarly treated young WI-38 cells. Nucleotide sequence analysis of this cDNA confirms its identity with that of a cDNA encoding a marker (p63) for the rough endoplasmic recticulum and a related swine hepatic cardiogenic shock protein. We show that LPC-1 expression in early PDL WI-38 cells is strictly cell cycle-regulated and its expression peaks 9–12 h after serum stimulation of G0cultures. The steady state levels of LPC-1 transcript in early PDL cells preceeding and following its peak expression are low, reflecting basal levels seen in G0upon removal of serum. Late PDL cells, however, seem to have lost this tight cell cycle regulation seen in early PDL cells and inappropriately express high levels of the transcript after serum stimulation. Specific antiserum detects a protein of approximately 63 kDa by Western analysis and elicits intense cytoplasmic staining of senescent fibroblasts by immunohistochemistry. Related genomic sequences are found in all mammalian species examined as well as in the chicken. These findings are consistent with the hypothesis that senescent WI-38 cells exhibit a state of growth arrest fundamentally distinct from that of quiescent (G0) young cells.

EGF-dependent phosphorylation of the EGF receptor in plasma membranes isolated from young and senescent WI-38 cells.

Tyrosine-specific phosphorylation of the receptor for epidermal growth factor (EGF) in plasma membranes isolated from WI-38 cells is EGF-dependent and occurs to an equivalent extent and on identical tryptic peptides in preparations from cells of various in vitro ages. There is a marked reduction, however, in phosphorylation of receptor molecules from senescent as compared with young WI-38 cells, if enzyme activity is assayed in an immune complex following solubilization of plasma membranes with Nonidet P-40 (NP-40). Differences in the level of receptor phosphorylation in young vs. senescent NP-40 extracts are not resolved by changing the temperature at which the assay is performed, or the length of incubation. Moreover, addition of NP-40 or chloroform-methanol extracts of young cells to assays measuring receptor phosphorylation in senescent cell NP-40 preparations does not augment the senescent enzyme activity. The immunopurified senescent receptor is, however, capable of catalyzing phosphorylation of exogenous substrates. These results indicate that the loss of receptor autophosphorylation in solubilized preparations may result from a differential sensitivity of the senescent cell receptor to the detergent. This finding provides a marker for senescence and suggests subtle changes in protein structure, conformation, or regulation of the EGF receptor in senescent cells.

Control of transcription in aging WI38 cells stimulated by serum to divide

In the present work, age-related alterations in histone acetylation and in the enzymatic system responsible for acetylation were examined in two growth states of WI38 fibroblasts. These observations were correlated with transcriptional activity of the cells when quiescent and after stimulation by serum to divide. In young WI38 cells (less than 50% lifespan completed), application of a growth stimulus resulted in sustained increase in transcription occurring after serum stimulation. However, senescent cultures (greater than 80% lifespan completed), while exhibiting 3-fold greater basal levels of transcription when quiescent, showed only 50% as great an increase in transcription following serum stimulation. Turnover of labile histone acetate was observed to decrease following stimulation of young cultures, but in senescent cultures, little turnover was evident either before or after stimulation. Examination of histone acetylase and deacetylase activity in isolated nuclei revealed that deacetylase activity was markedly altered between the resting and growing state and also with age. In resting young cells, two rates of deacetylase activity were apparent, but stimulated cells exhibited only one rate. In addition, at the end of the reaction period, resting cells had removed twice as much incorporated [ 14C]acetate as had stimulated cells. In contrast, both stimulated and quiescent senescent fibroblasts exhibited two rates of deacetylation, but little deacetylase activity was apparent in either case. The present studies demonstrate an inverse correlation between rates of RNA synthesis and turnover of histone acetate. Enzymatic studies suggest that this result is due to a decrease in histone deacetylase activity.

Extension of the lifespan of cultured normal human diploid cells by vitamin E.

Inclusion of vitamin E (DL-alpha-tocopherol) in the culture medium for human diploid cells greatly prolongs their in vitro lifespan. The addition of 100 mug of DL-alpha-tocopherol per ml of medium has allowed us to culture WI-38 cells for more than 100 population doublings to date. (These cells normally have an in vitro lifespan of 50 +/- 10 population doublings.) Cells at the 100th population doubling have a normal diploid karyotype, appear to behave in all other respects like young WI-38 cells, and are still actively dividing. We interpret this result as support for the free radical theory of aging.