Glucose restriction can extend normal cell lifespan and impair precancerous cell growth through epigenetic control of hTERT and p16 expression

Abstract

Cancer cells metabolize glucose at elevated rates and have a higher sensitivity to glucose reduction. However, the precise molecular mechanisms leading to different responses to glucose restriction between normal and cancer cells are not fully understood. We analyzed normal WI-38 and immortalized WI-38/S fetal lung fibroblasts and found that glucose restriction resulted in growth inhibition and apoptosis in WI-38/S cells, whereas it induced lifespan extension in WI-38 cells. Moreover, in WI-38/S cells glucose restriction decreased expression of hTERT (human telomerase reverse transcriptase) and increased expression of p16(INK4a). Opposite effects were found in the gene expression of hTERT and p16 in WI-38 cells in response to glucose restriction. The altered gene expression was partly due to glucose restriction-induced DNA methylation changes and chromatin remodeling of the hTERT and p16 promoters in normal and immortalized WI-38 cells. Furthermore, glucose restriction resulted in altered hTERT and p16 expression in response to epigenetic regulators in WI-38 rather than WI-38/S cells, suggesting that energy stress-induced differential epigenetic regulation may lead to different cellular fates in normal and precancerous cells. Collectively, these results provide new insights into the epigenetic mechanisms of a nutrient control strategy that may contribute to cancer therapy as well as antiaging approaches.

Figure 1

Glucose restriction inhibits proliferation of WI-38/S cells but not of WI-38 cells. A) Graphic presentations of growth kinetics of WI-38 cells (left panel) and WI-38/S (right panel) after either 12- or 4-wk culture periods, respectively, with or without glucose-restricted growth medium. Viable cells were counted weekly by trypan blue staining using a hemacytometer. Cell growth rates were calculated by the formula N/N₀, where N = number of cells in growth vessel at end of growth period and N₀ = number of cells plated in growth vessel. Results were obtained from 3 independent experiments. Error bars = se. B) Morphological changes of WI-38 cells (left panels) and WI-38/S (right panels) were observed at 6 and 4 wk of treatment, respectively, with or without glucose-restricted medium. View, ×100.

Figure 2

Glucose restriction results in apoptosis in WI-38/S cells but not WI-38 cells. A) Cell apoptosis of WI-38 (left panel) and WI-38/S cells (right panel) was detected by using an annexin V and PI staining system following the manufacturer‘s instructions (Invitrogen). A BD FACSCalibur apparatus was used to acquire and analyze a minimum of 10⁵ events using the CellQuest program. Cells were harvested and analyzed for apoptosis weekly. Graphs are representative of similar results obtained from 3 independent experiments. B) Histogram of the apoptosis rate in WI-38 and WI-38/S cells in response to glucose restriction. Error bars = se. *P < 0.05 vs. normal glucose control.

Figure 3

Glucose restriction (GR) results in expression alterations of the hTERT and p16 genes. A) Graphic presentation of relative mRNA levels of hTERT (left panel) and p16 (right panel) in WI-38 and WI-38/S cells within 2- and 4-wk treatment periods. WI-38 and WI-38/S cells were cultured in regular or glucose-restricted medium as indicated previously. Cell pellets were collected weekly and subjected to quantitative real-time PCR analysis. Data are in triplicate from 3 independent experiments and were normalized to GAPDH and calibrated to levels in samples with normal glucose medium. Error bars = se. *P < 0.05 vs. normal glucose control. B) Protein levels of p16 were determined by Western blot analysis. Cell proteins were extracted and 50 μg of protein was resolved on 4–12% SDS-PAGE, transferred onto nitrocellulose membrane, and probed with monoclonal p16 antibody. Membranes were reprobed with anti-actin antibody to ensure for equal loading. Photographs are representative of an experiment that was repeated 3 times.

Figure 4

Histone modification changes of the hTERT and p16 promoters in response to glucose restriction. A) Glucose restriction-treated and untreated WI-38 and WI-38/S cells were analyzed by ChIP assays using chromatin markers including acetyl-H3, acetyl-H4, HDAC1, dimethyl-H3K4, and trimethyl-H3K9 and no antibody controls in the promoter region of hTERT (left panel) and p16 (right panel). PCR primers and conditions were used as described in Materials and Methods. Photographs are representative of an experiment that was repeated in triplicate. B) Histone modification enrichment of hTERT (left panel) and p16 (right panel) was calculated from the corresponding DNA fragments amplified by PCR as described previously. Error bars = se.

Figure 5

Glucose restriction-induced methylation alteration of p16 promoter regions in normal WI-38 cells. A) CpG density in the p16 promoter region. B) Methylation status of the p16 promoter in glucose restriction-treated and untreated WI-38 cells was determined by direct bisulfite sequencing analysis. Bisulfite sequencing primers and conditions were used as described in Materials and Methods. Horizontal rows represent individual CpG sites from the region −282 to +10 of the p16 promoter. Methylation status at CpG dinucleotides: ○, unmethylated cytosine; •, methylated cytosine; , partial methylated cytosine. C) Sequencing at ∼−190 of the p16 promoter, which is the putative E2F-1 binding site. Arrows indicate changed CpG sites of the E2F-1 binding site. D) Chromatin DNA from glucose restriction-treated and untreated WI-38 cells was immunoprecipitated by E2F-1 antibody together with mouse IgG controls. Photograph is representative of an experiment that was repeated in triplicate. E) Schematic presentation of E2F-1 blocking access to the methylated E2F-1 binding site in the p16 promoter in response to glucose restriction.

Figure 6

Effects of epigenetic modulators on p16 and hTERT in response to glucose restriction. 5-Aza- or TSA-treated and untreated WI-38 and WI-38/S cells in regular glucose or glucose-restricted (GR) medium were transiently transfected with p16, hTERT, or basic pGL-2 luciferase plasmids together with Renilla luciferase (pRL-SV40) plasmids as the internal controls for 24 h in normal or glucose-restricted growth medium. A, D) Untreated WI-38 and WI-38/S cells. Luciferase activity in control samples transfected with basic plasmid pGL-2 was normalized to 1.0. B, E) 5-Aza-treated WI-38 and WI-38/S cells. Cells were treated with 2.5 μM 5-aza for 48 h and underwent transfection as described previously. 5-Aza-untreated p16 or hTERT samples were normalized to 1.0. C, F) TSA-treated WI-38 and WI-38/S cells. After 24 h of treatment with 100 ng/ml TSA, WI-38 and WI-38/S cells were transiently transfected with plasmids as indicated previously. TSA-untreated p16 or hTERT samples were normalized to 1.0. Values are means ± se of 3 independent experiments.

Figure 7

Alterations of DNMT and HDAC enzymatic activities in response to glucose restriction. Nuclear proteins of WI-38 and WI-38/S cells were extracted at the indicated time points as described in Materials and Methods. DNMT (left panel) and HDAC (right panel) activity assays were performed according to the manufacturer’s protocols. Values are means ± se of 3 independent experiments. *P < 0.05 vs. normal glucose control.