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FASEB J. May 2010; 24(5): 1442–1453.
PMCID: PMC2996891

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

Yuanyuan Li,* Liang Liu, and Trygve O. Tollefsbol*§‖,1

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 p16INK4a. 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.—Li, Y., Liu, L., Tollefsbol, T. O. Glucose restriction can extend normal cell lifespan and impair precancerous cell growth through epigenetic control of hTERT and p16 expression.

Keywords: cancer, DNA methylation, histone modification, E2F-1, longevity

Caloric restriction has been considered a potent physiological approach to cancer prevention and therapy for several decades. The different responses in the consumption and metabolism of glucose, the major caloric source in the human body, between cancer and normal cells could be a promising cancer preventive and/or therapeutic target (1,2,3). The metabolism of glucose has attracted extensive interest in changes of glycolysis in cancer cells, which is known as the Warburg effect (4). Alterations in the tumor microenvironment, which is characterized by regions of fluctuating and chronic hypoxia and low extracellular pH contribute significantly to tumor progression (5). Glucose restriction is a metabolic stressor that triggers several signal transduction pathways (6). The sensitivity to glucose in many cancer cells has been used successfully for cancer diagnosis and monitoring (7). Furthermore, glucose restriction results in a cellular stress-induced multiple gene expression alteration response, which includes many cell growth and survival-related genes (8, 9). However, the mechanisms by which glucose restriction exerts its effect on carcinogenic processes are only beginning to be elucidated.

Nutrition is believed to be a chief contributor to the regulation of gene expression in both physical and pathological processes by affecting epigenetic pathways, which include two basic processes: DNA methylation and histone modification (10,11,12,13). Furthermore, caloric restriction-induced prolongation of lifespan in various organisms has been shown to be partly related to the NAD+-dependent histone deacetylase family member, Sirt-1, which is involved in a wide variety of cellular processes, including aging and stress response regulated by epigenetic processes (14). Therefore, epigenetic-mediated changes in gene expression in response to glucose restriction may be a major molecular mechanism linking environmental factors with consequences for cell function in normal and cancer cells.

A key determinant of the enzymatic activity of human telomerase, hTERT (human telomerase reverse transcriptase), has drawn extensive interest recently because its up-regulated expression is present in ~90% of malignant tumors but is barely detectable in normal somatic cells (15, 16). Another cell cycle regulator gene, p16INK4a, is also believed to play a crucial role in tumor growth suppression and cell senescence (17, 18). Notably, both hTERT and p16 are epigenetics-sensitive genes, in that their expression is frequently regulated by epigenetic processes (19, 20). Therefore, focusing on the epigenetic modulation of the expression of these two key genes can facilitate elucidation of the influences of epigenetic mechanisms either on normal cells or on cancer cells that have undergone glucose reduction.

To elucidate the role of epigenetic control in normal and cancer cells in response to glucose restriction, we used fetal lung fibroblast WI-38 cells and immortalized WI-38 (WI-38/S) cells, which were derived from WI-38 cells by transfection with simian virus-40 (SV-40) antigen. Analyses of these two types of cells, which exhibit normal and precancerous characteristics, respectively, but share the same origin, provide an opportunity to assess the mechanisms by which the effects of glucose reduction are exerted to influence gene expression through epigenetic regulation. In the current study, we found that glucose reduction induced growth inhibition and apoptosis in the immortalized cells but not in the normal cells. This result is due, at least in part, to differential modulation of hTERT and p16 expression through DNA methylation changes and/or histone remodeling in normal and immortalized cells in response to glucose restriction. Our findings not only reveal epigenetic mechanisms of caloric restriction on cancer development but also provide new insights into nutrition-related cancer prevention and therapy.

MATERIALS AND METHODS

Cell culture and growth kinetics assessment

Normal WI-38 diploid human lung fibroblasts were obtained from American Type Culture Collection (Manassas, VA, USA), and WI-38 immortalized cells (WI-38/S) were derived from WI-38 cells that were stably transfected with retrovirus as described previously (21). Cells were maintained in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 4.5 g/L glucose. To restrict glucose, cells were cultured in glucose- and pyruvate-free DMEM (Invitrogen). All culture media were supplemented with 10% FBS (Atlanta Biologicals, Lawrenceville, GA, USA) and 1% penicillin/streptomycin (Mediatech, Herndon, VA, USA) in the presence of 5% CO2 at 37°C. The actual glucose concentration in glucose restriction medium is 15 mg/L, which was assessed by a glucose assay kit (Biovision, Mountain View, CA, USA) following the manufacturer’s protocols. Normal WI-38 cells were used at the start of passage 15. Cells were passaged weekly at a seeding density of 105 cells/plate and counted using a Neubauer hemocytometer. The cell growth kinetics were calculated by the following formula: growth rate = N/N0 (where N is the number of cells in the growth vessel at the end of a period of growth, and N0 is the number of cells plated in the growth vessel). Morphological changes in the cells were recorded using a Nikon Coolpix990 digital camera (Nikon, Tokyo, Japan).

Retroviral transduction

The retroviral vector pBabe-neo-SV/40 T/t antigen and the construct expressing vesicular stomatitis virus G-glycoprotein were kindly provided by Dr. Robert Weinberg (Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA, USA) (22) and Dr. Chris Klug (Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA), respectively. Retroviruses were produced and used to infect Phoenix gag-pol cells as described previously (21). WI-38 cells were infected with amphotropic retroviral supernatants and were selected with G418 (400 μg/ml) (Sigma-Aldrich Corp., St. Louis, MO, USA) for 10 d, and the cell clones obtained were pooled. The infected WI-38 cells attained immortalized status and were subsequently treated with glucose-restricted medium at population doubling 50 as described previously (21).

Cell apoptosis analysis

Normal WI-38 and immortalized WI-38/S cells with or without glucose restriction treatment were collected and washed with cold PBS weekly. Cells were then used for apoptosis analysis with the Vybrant Apoptosis Assay Kit 2 (Invitrogen). After fixation with annexin-binding buffer, cells were stained with both Alexa Fluor annexin V and propidium iodide (PI) according to the manufacturer’s instructions. Flow cytometry analyses were performed on a FACSCalibur flow cytometer (BD, Franklin Lakes, NJ, USA). The fluorescence intensity of the viable cells was analyzed using CellQuest software (BD).

Reporter gene assay of promoter activity

The pGL-2 luciferase vectors containing p16 and hTERT promoter constructs were kindly provided by Dr. Gordon Peters (Imperial Cancer Research Fund Laboratories, London, UK) and Dr. Silvia Bacchetti (Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada), respectively (23, 24). Before transfection, normal WI-38 and immortalized WI-38/S cells were grown in 24-well culture plates in normal or glucose-restricted medium. For epigenetic regulator treatment, attached cells were treated with 2.5 μM DNA methyltransferase (DNMT) inhibitor, 5-aza-2′-deoxycytidine (5-aza) (Sigma-Aldrich), and 100 ng/ml histone deacetylase (HDAC) inhibitor, trichostatin-A (TSA) (Sigma-Aldrich), for 48 and 24 h, respectively. The medium with 5-aza and TSA was replaced every 24 h for the duration of the experiment. Cells were then transiently transfected with either p16 or hTERT promoter-luciferase constructs along with pGL-2 vector (Promega Corp., Madison, WI, USA) as a basic control for 24 h, respectively. Luciferase activity was measured in cell lysates by a microplate luminometer using a Dual Luciferase Assay Kit (Promega) according to the manufacturer’s protocol. Luciferase activity was normalized by Renilla luciferase activity through cotransfection with the pRL-SV40 plasmid (Promega). Each experiment was repeated in triplicate.

DNMT and HDAC activities

Cultured WI-38 and WI-38/S cells were harvested at the indicated time points as described above, and nuclear extracts were prepared with nuclear extraction reagent (Pierce Biotechnology, Rockford, IL, USA). The DNMT (Epigentek, Brooklyn, NY, USA) and HDAC (Upstate Biotechnology, Charlottesville, VA, USA) activity assays were performed according to the manufacturer’s protocols. The enzymatic activities of DNMTs and HDACs were detected by a microplate reader at 450 nm and fluorescent plate reader at excitation/emission of 353/448 nm, respectively.

Quantitative real-time PCR

Both WI-38 and WI-38/S cells were cultured and treated as described above. Total RNA was extracted using an RNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Genes of interest were amplified using 5 μg of total RNA reverse-transcribed to cDNA using a Superscript II kit (Invitrogen) with oligo(dT) primer. In the real-time PCR step, PCR reactions were performed in triplicate with 1 μl cDNA/10 μl reaction and primers specific for hTERT (Hs00162669_ml), p16 (Hs00923894_ml), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Hs99999905_ml) provided by Inventoried Gene Assay Products (Applied Biosystems, Foster City, CA, USA) using the Platinum Quantitative PCR Supermix-UDG (Invitrogen) in a Roche LC480 thermocycler (Roche Diagnostics, Basel, Switzerland). Thermal cycling was initiated at 94°C for 4 min followed by 35 cycles of PCR (94°C for 15 s and 60°C for 30 s). GAPDH was used as an endogenous control, and vehicle control was used as a calibrator. The relative changes in gene expression were calculated using the following formula: fold change in gene expression, 2−ΔΔCt = 2−{ΔCt(GT) ΔCt(GNT)}, where ΔCt = Ct (hTERT or p16) − Ct (GAPDH), GT represents glucose treatment samples, GNT represents glucose no-treatment control samples, and Ct represents threshold cycle number.

Western blot analysis

For Western blot analysis, protein extracts were prepared by TRAPeze 1× CHAPS cell lysis buffer (Chemicon International, Temecula, CA) from normal and immortalized WI-38 cells in normal or glucose-restricted medium according to the manufacturer’s protocol. Proteins (50 μg) were electrophoresed on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were probed with monoclonal antibody to p16INK4a (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and then the membrane was stripped and reprobed with GAPDH antibody (V-18, Santa Cruz Biotechnology) as loading control. Molecular weight markers were run on each gel to confirm the molecular size of the immunoreactive proteins. Immunoreactive bands were visualized using the enhanced chemiluminescence detection system (Santa Cruz Biotechnology) following the protocol of the manufacturer.

Chromatin immunoprecipitation (ChIP) assay

WI-38 and WI-38/S cells were cultured in 100-mm tissue culture dishes and harvested at the indicated time points. ChIP assays were performed with an EZ ChIP assay kit according to the manufacturer’s protocol (Upstate Biotechnology) as described previously (10, 11). The antibodies used in the ChIP assays were ChIP-validated acetyl-histone H3 (Upstate Biotechnology), acetyl-histone H4 (Upstate Biotechnology), HDAC1 (Santa Cruz Biotechnology), dimethyl-histone H3 (Lys4) (Upstate Biotechnology), and trimethyl-histone H3 (Lys9) (Upstate Biotechnology), respectively. ChIP-purified DNA was amplified by standard PCR using primers specific for the hTERT and p16 promoters. The hTERT ChIP primers were sense 5′-CAGGACCGCGCTTCCCACG-3′ and antisense 5′-GGCTTCCCACGTGCGCAGC-3′, rendering a 236-bp PCR product. The p16 ChIP primers were sense 5′-TAGGAAGGTTGTATCGCGGAGG-3′ and antisense 5′-CAAGGAAGGAGGACTGGGCTC-3′, rendering a 172-bp PCR product. PCR amplification was performed using 2XPCR Master Mix (Promega Corp.), and the reaction was initiated at 94°C for 4 min, followed by 30 cycles of PCR (94°C for 30 s, 56°C for 30 s, and 72°C for 1 min), and extended at 72°C for 5 min. After amplification, PCR products were separated on 1.5% agarose gels and visualized by ethidium bromide fluorescence using Kodak 1D 3.6.1 image software (Eastman Kodak Co., Rochester, NY, USA). Quantitative data were analyzed using Sequence Detection System 2.1 software (PE Applied Biosystems, Foster City, CA, USA).

Bisulfite sequencing analysis

To assess the methylation status changes in the hTERT and p16 promoters, sodium bisulfite methylation sequencing was performed as described previously (10, 11). Approximately 1 μg of DNA was treated with bisulfite following the manufacture’s protocol (Human Genetic Signatures, Macquarie Park, NSW, Australia). Bisulfite-modified DNA was analyzed using sequencing analysis of the promoter region of the hTERT and p16 genes. The bisulfite primers for hTERT and p16 were sense 5′-GGTTTTTAGTGGATT-3′ and antisense 5′-AAAACCAAAACTTCCCAC-3′ and sense 5′-GATTTTAGGGGTGTTATATT-3′ and antisense 5′-AAAACTCCATACTACTCCCC-3′, respectively. For sequencing analysis, PCR products were purified using a gel extraction kit (Qiagen) and were directly sequenced on an automated DNA sequencer. Each sample was sequenced three times to determine the site-specific methylation changes in the amplified regions as described previously (10, 11).

Statistical analyses

Results for real-time PCR and luciferase assays were derived from at least three independent experiments. Kodak 1D 3.6.1 image software was used for quantification of ChIP PCR products. Statistical significance between treatment and control groups was evaluated using Student’s t test, and values are presented as the mean ± se. P < 0.05 was considered statistically significant.

RESULTS

Glucose restriction results in an extended lifespan of WI-38 cells and reduced survival of immortalized WI-38/S cells

To determine the long-term effects of glucose reduction on normal WI-38 and immortalized WI-38/S cells, we performed cell growth assays and morphological analyses to detect cell proliferation status. As shown in Fig. 1 (right panels), decreased proliferation capacity and subsequent cell death were clearly observed by 4 wk in WI-38/S cells in glucose-reduced medium compared with the cells in normal medium. However, this process did not interfere with normal WI-38 cells. The morphology of WI-38 cells in glucose-reduced medium was also displayed normally with increasing culture time (Fig. 1, left panels). In WI-38 cells, a slightly reduced growth rate was observed in the first 4 wk of treatment with glucose restriction compared with WI-38 cells in normal glucose medium. After that, the situations switched between these two conditions and, more importantly, glucose-restricted WI-38 cells underwent a total expansion of lifespan by more than 4 wk (Fig. 1A, left panel). These results indicate that glucose restriction inhibits cell proliferation in WI-38/S cells more than that in normal WI-38 cells, suggesting that immortalized WI-38/S cells exhibit a higher sensitivity to glucose reduction. Furthermore, the results of the lifespan elongation in glucose-restricted normal WI-38 cells are consistent with the studies on caloric restriction and longevity in human populations (25), indicating that glucose deficiency can also induce longevity in normal cells.

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 restriction-induced apoptosis occurred in WI-38/S cells but not in WI-38 cells

To address how glucose restriction affects the proliferation of the cells, we performed cell apoptosis assays on normal WI-38 and immortalized WI-38/S cells with the aforementioned treatment. We found that glucose restriction resulted in significant apoptosis at 17.7% in immortalized WI-38/S cells compared with 1.77% of the WI-38/S cells cultured in the normal glucose medium, as indicated in Fig. 2. However, much lower levels of apoptosis were detected in normal WI-38 cells both in regular (0.39%) and glucose-restricted medium (1.27%), which matched the normal range of apoptosis rate. Consistent with the results shown in Fig. 1, these findings indicate that glucose restriction significantly induces apoptosis in immortalized WI-38/S cells but has little if any effect on normal WI-38 cells and that this property could have implications for cancer preventive and/or therapeutic approaches.

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 ...

Glucose reduction causes hTERT suppression and p16 activation in WI-38/S cells

Considering the different sensitivity to glucose restriction between the normal WI-38 and precancerous WI-38/S cells, we next attempted to sort out what mechanisms and genes may underlie these phenomena. It is well known that high activity of telomerase is present in the majority of cancer cells, which allows the cells to survive and proliferate. hTERT, the key enzymatic component of telomerase, is up-regulated in most tumor cells. We also focused on an important tumor suppressor gene, p16, in this study because of its crucial role in regulation for cell proliferation and senescence. These properties of the hTERT and p16 genes prompted us to investigate the expression alteration of these genes in response to glucose restriction in normal WI-38 and immortalized WI-38/S cells. To investigate the effects of glucose restriction on hTERT and p16 expression, we performed real-time PCR to analyze these alterations. As illustrated in Fig. 3A (left panel), we found that glucose restriction significantly increased hTERT transcription by 3.2- and 4.9-fold in normal WI-38 cells (P<0.05), whereas it decreased hTERT transcription in WI-38/S cells by 6.25- and 2.63-fold (P<0.05) in 2- and 4-wk treatments compared with cells grown in the regular glucose medium. These results indicated that hTERT expression may play an important role in modulation of differential sensitivity to glucose reduction in normal and precancerous cells. However, these effects were less pronounced in WI-38/S cells for the 4-wk treatment compared with the 2-wk treatment of glucose reduction, indicating that cancer cells may elicit some alternative mechanisms to overcome the nutrition stress when they undergo long-term glucose deficiency. As shown in Fig. 3A (right panel), we also found that glucose reduction could gradually induce down-regulation of p16 expression by 4- and 7.1-fold (P<0.05) in 2- and 4-wk treatments, respectively, in normal WI-38 cells. However, p16 expression was up-regulated in WI-38/S cells in response to glucose restriction. We also observed alterations of p16 protein levels in response to glucose restriction in WI-38 and WI-38/S cells. As indicated in Fig. 3B (left panel), in normal glucose-treated WI-38 cells p16 expression was increased in the regular growth medium, whereas no p16 protein signal was detected in glucose restriction medium for the rest of the experiment. However, glucose restriction increased p16 expression in immortalized WI-38/S cells as shown in Fig. 3B (right panel), which was consistent with our previous real-time PCR results. These results indicated that glucose restriction affected hTERT and p16 expression in an opposite manner between the normal and precancerous cells, which could lead to different cell fates, that is, either extending the cell lifespan in normal WI-38 cells or triggering cell apoptosis and/or senescence in precancerous WI-38/S cells.

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 ...

Glucose restriction results in chromatin remodeling of the promoters of the hTERT and p16 genes

Previous studies have shown that both the hTERT and p16 genes are epigenetics-sensitive and their expression is often modulated by epigenetic processes. We therefore investigated the molecular mechanisms of the aforementioned effects induced by glucose restriction on epigenetic processes in normal WI-38 and immortalized WI-38/S cells. In addition to DNA methylation as the primary mechanism of epigenetic control, histone acetylation and/or methylation also play important roles in gene regulation. Furthermore, HDACs are known transcription repressors that can recruit repressor complexes to target gene promoters and were therefore used as an epigenetic marker in this study. To explore whether these chromatin markers affect hTERT and p16 gene expression in response to glucose restriction, we performed ChIP assays of the hTERT and p16 genes in WI-38 and WI-38/S cells.

We analyzed the hTERT and p16 promoters by using antibodies for both transcriptionally active (acetyl-H3, acetyl-H4, and dimethyl-H3K4) and inactive (trimethyl-H3K9 and HDAC1) markers of chromatin. The results indicated that glucose restriction increased the active chromatin markers including acetyl-H3, acetyl-H4, and dimethyl-H3K4 in the hTERT promoter by 3.18-, 2.65-, and 8-fold, respectively, whereas it decreased trimethyl-H3K9 and HDAC1, the inactive chromatin markers, by 1.16- and 20-fold, respectively, compared with the normal glucose controls in normal WI-38 cells (Fig. 4, left panel). However, we found opposite results in the immortalized WI-38/S cells in which the inactive chromatin markers were enriched in the hTERT promoter, whereas the active chromatin markers declined in response to glucose restriction as indicated in Fig. 4, left panel. Consistent with our previous studies on different patterns of hTERT expression in normal WI-38 and immortalized WI-38/S cells, these results suggest that glucose restriction may result in different cell fates by influencing hTERT transcription regulation through chromatin modification primarily through alterations of acetylation and methylation in specific histone residues in the hTERT promoter. However, H3 trimethylation at lysine 9, a repressive chromatin marker, changed very little in the hTERT promoter for both WI-38 and WI-38/S cells with glucose restriction, suggesting that histone methylation of this specific residue may play less of a role in glucose reduction-mediated hTERT regulation.

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, ...

We further examined chromatin modifications in the promoter of p16 and found a prominent decrease of active chromatin markers in the p16 promoter in normal WI-38 cells with glucose restriction, indicating that glucose restriction-induced p16 down-regulation in WI-38 cells may be due to epigenetic control through histone modification (Fig. 4, right panel). In contrast, in WI-38/S cells, glucose reduction resulted in enrichment of active chromatin markers in the p16 promoter, which correlated with the increased levels of p16 mRNA expression leading to apoptosis and senescence in precancerous WI-38/S cells. However, we did not find obvious changes in HDAC1 binding to the p16 promoter in WI-38/S cells, suggesting that a potential alternative pathway may be involved in histone acetylation regulation beyond HDAC1 control in precancerous cells in response to glucose restriction. Collectively, these results suggest that the different consequence in normal and precancerous cells in response to energy stress such as glucose reduction may be due to the reactive gene expression changes (hTERT and p16) through epigenetic regulation involving chromatin modification.

DNA methylation status of hTERT and p16 promoters in response to glucose deficiency

Because DNA methylation plays an important role in gene expression regulation, we analyzed the DNA methylation status of the hTERT and p16 promoters by performing bisulfite methylation sequencing analysis. To elucidate the effects of methylation on the hTERT promoter, we analyzed the methylation status of the hTERT promoter in the region −298 to −31, which contains many CpG dinucleotides and overlapping transcription factor binding sites. However, there was no methylation status change in the hTERT promoter in either WI-38 and WI-38/S cells with or without glucose restriction treatment (data not shown), which indicated that DNA methylation may not play a role in glucose restriction-mediated altered hTERT expression. In addition, we also assessed the methylation status of the p16 promoter around region −282 to +10, as shown in Fig. 5A. In normal WI-38 cells, we found increased DNA methylation around region −190 of the p16 promoter, which was identified as a putative binding site of E2F-1, an active transcription factor of p16 (26), with 4 wk of glucose restriction treatment compared with 2 wk of treatment as shown in Fig. 5B. However, the methylation level of the p16 promoter in WI-38/S cells did not show differences between 2 and 4 wk of cell culture periods (data not shown). It has been extensively reported that E2F-1 is one of the most important transcription factors of the INK4 family with methylation-sensitive properties and its binding is affected by the methylation status of its recognition sites in promoter regions (27). To further elucidate the effects of methylation on transcription factor binding to the p16 promoter, we performed ChIP assays and found that the binding of E2F-1 to the p16 promoter was increased in normal glucose medium compared with glucose restriction medium as indicated in Fig. 5D. Interestingly, our results also indicate that glucose restriction-induced hypermethylation of an E2F-1 binding site blocks E2F-1 access to the p16 promoter and contributes to p16 down-regulation, which may lead to escape from apoptosis and senescence processes and extend lifespan in normal WI-38 cells (Fig. 5E).

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 ...

Effects of epigenetic modulators on p16 and hTERT expression in response to glucose restriction

To further explore the potential epigenetic mechanisms involving differential expression patterns of p16 and hTERT in normal and immortalized WI-38 cells induced by glucose restriction, we have introduced two epigenetic regulators into this study including the DNMT inhibitor, 5-aza, and the HDAC inhibitor, TSA. By using the luciferase reporter assay (Fig. 6A, D), we found that glucose restriction can inhibit p16 promoter activity and increase hTERT promoter activity in normal WI-38 cells. In contrast, however, p16 promoter activity was increased and hTERT promoter activity was decreased in response to glucose restriction in immortalized WI-38/S, which was consistent with our previous studies.

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 ...

We therefore treated cells with two epigenetic modulators, 5-aza and TSA, and found that the promoter activities of p16 and hTERT can be efficiently regulated by 5-aza and TSA in normal WI-38 cells, especially in a situation of glucose restriction as shown in Fig. 6B, C, E, F (left panel). For example, the promoter activity of p16 was reactivated owing to hypomethylation of the p16 promoter induced by 5-aza in WI-38 cells. Furthermore, this reactivation of p16 is more prominent in glucose-restricted WI-38 cells compared with cells in normal glucose medium (Fig. 6B, left panel). However, in immortalized WI-38/S cells, we found that the promoter activities of p16 and hTERT in glucose-restricted WI-38/S cells were less responsive to 5-aza treatment compared with cells in normal glucose medium (Fig. 6B, E, right panels). Moreover, TSA treatment did not affect the promoter activities of p16 and hTERT either in normal glucose or glucose-restricted WI-38/S cells (Fig. 6C, F, right panels). These results indicate that epigenetic mechanisms play a more important role in regulation of p16 and hTERT expression in normal WI-38 than in WI-38/S cells in response to glucose restriction.

Alterations of epigenetic enzymatic activity in response to glucose restriction

To further interpret the epigenetic modulations on p16 and hTERT expression in response to glucose restriction in normal and immortalized WI-38 cells, we also assessed epigenetics-related enzymatic activities including DNMT and HDAC activities. As indicated in Fig. 7, in normal WI-38 cells, both DNMT and HDAC activities were activated by 4.48- and 1.66-fold, respectively, in response to glucose restriction. The significantly increased DNMT activity in glucose-restricted WI-38 cells may contribute to hypermethylation of the p16 promoter, resulting in p16 repression as shown in Fig. 5. However, the enzymatic activation of DNMTs and HDACs was less pronounced (by 1.21- and 1.23-fold, respectively) in glucose-restricted WI-38/S cells than in normal WI-38 cells. These results suggest that the normal cells can respond well to energy stress by regulating a series of gene expressions such as p16 and hTERT that have favorable effects on cellular growth through triggering of epigenetic mechanisms. However, metabolic stress in cancer cells seems to lead to aberrant gene expressions such as p16 accumulation and hTERT down-regulation due to decreased effects of epigenetic regulation. In summary, differential modulation of epigenetic mechanisms induced by energy stress in normal and immortalized cells causes differential gene expression patterns, leading to various cellular fates such as apoptosis, senescence, and cellular lifespan extension.

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) ...

DISCUSSION

Evidence has accumulated that glucose restriction may play an important role in cancer prevention and therapy, and there is emerging interest in identifying the basic mechanisms underlying this phenomenon. In the present study, we focused on the molecular mechanisms of glucose restriction-induced cell senescence and apoptosis both in normal WI-38 fetal lung fibroblast cells and in immortalized WI-38/S cells. In particular, our results demonstrate that glucose restriction induces apoptosis in precancerous cells but not in normal WI-38 cells and the different response among the cells may be partially due to the variable expression levels of hTERT and p16 regulated by epigenetic factors. Therefore, our studies facilitate an approach to cancer prevention and therapy by controlling glucose intake and metabolism and may also provide insights into crucial effects of epigenetic regulation under certain stresses such as glucose restriction.

It has been extensively reported that cancer cells are more sensitive to glucose concentration than are normal cells because of a higher consumption ratio of energy in cancer cells (28, 29). To explore the effects of glucose restriction on normal and cancerous cells, in this study we chose WI-38 and SV-40 antigen-transfected immortalized WI-38 cells to represent normal and precancerous cells. Because these cells share the same derivation, comparison of these two types of cells yields a unique benefit in this study. We found that in normal WI-38 cells glucose restriction extended the cellular lifespan by an extra 4 wk compared with the cells grown in normal glucose medium, which is consistent with results of previous studies showing extended longevity caused by caloric restriction both in experimental animal models and in the human body (25, 30). However, we observed opposite results in immortalized WI-38/S cells, which underwent apoptosis in glucose-reduced medium, whereas the cells in normal glucose medium maintained high proliferative rates. Several mechanisms were proposed previously to explain how glucose restriction causes apoptosis in cancer rather than normal cells, including different requirement levels of ATP, protein synthesis, glutathione, or nucleotides, as well as changes in the cellular redox state (31, 32). However, the precise molecular mechanisms still remain unknown.

There has been increasing interest in the effects of epigenetic mechanisms on gene expression regulation both in physiological and pathological processes. These epigenetic alterations involve both losses and gains of DNA methylation as well as altered patterns of histone modifications (12, 33). In the present study, we focused on two crucial genes, hTERT and p16, not only because these genes play important roles in tumor development and aging processes but also because the transcription regulation of these genes is sensitive to changes in epigenetic processes such as histone modification and DNA methylation (19, 20).

In the current study, we found that glucose restriction increased hTERT expression and decreased p16 expression and thus promoted proliferation and longevity in normal WI-38 cells. However, a decrease in hTERT expression and increase in p16 expression induced by glucose restriction contributes to apoptosis in immortalized WI-38/S cells. p16 is primarily associated with cell cycle arrest, although up-regulated p16 can potentiate apoptosis processes through indirect signal pathways induced by certain oncogenes such as c-myc (17). Moreover, decreased hTERT and telomerase activities help to trigger apoptotic caspase by activation of a series of apoptosis signal pathways (34). These results indicate that the transcription regulation of hTERT and p16 during glucose reduction may play an important role in leading to different cell fates such as proliferation and apoptosis in normal and precancerous cells. hTERT is the key determinant of the enzymatic activity of human telomerase, which has increased expression in stem cells, cancer-derived cell lines, and spontaneously immortalized cells in culture and is detectable in ~90% of tumors (15, 16). Multiple mechanisms involved in hTERT gene transcription regulation, which include cellular and viral oncogenic factors as well as the epigenetic pathways, have been proposed (19, 35). Recently, we demonstrated that a dietary component, genistein, can repress hTERT expression via epigenetic mechanisms, suggesting that hTERT expression could be flexibly regulated by environmental factors (10). The p16 gene is a crucial tumor suppressor gene and the silenced expression of p16 is believed to involve aberrant epigenetic regulation in cancer cells (17). Epigenetic changes occur frequently in response to multiple physical and pathological stresses including energy deficiency and tumor development. We therefore sought to explore the mechanisms underlying the transcription regulation of hTERT and p16 in normal and precancerous cells in response to glucose restriction through detection of the epigenetic status for these two genes.

Epigenetic processes involving chromatin modification, such as histone acetylation, methylation, and phosphorylation, are believed to play important roles in controlling gene transcription (12, 13). We further investigated chromatin remodeling pathways as mechanisms for effects of glucose restriction on gene expression through ChIP analysis. Our results showed that glucose restriction led to an increase in the acetylation of histones H3 and H4 as well as dimethyl-H3K4 (active markers of chromatin) and a decrease in HDAC1 and trimethyl-H3K9 (suppressive markers) around the transcription start site of hTERT, whereas opposite results were observed in the p16 promoter, leading to up-regulated expression of hTERT and down-regulated p16 in normal WI-38 cells. Similar results were observed in immortalized WI-38/S cells except for HDAC1 binding to the p16 promoter, suggesting that loss of HDAC1 enrichment may not be the only cause of histone acetylation recruitment in the promoter region of p16 in response to glucose restriction. These findings suggest that altered hTERT and p16 gene expression by glucose restriction is due at least in part to alterations in chromatin modification. More important, the expression alterations of the hTERT and p16 genes may not only result in apoptosis in precancerous cells but also favor the normal cells undergoing longevity extension through an influence on epigenetic processes.

In addition to chromatin modifications, DNA methylation, acting as a major epigenetic modulator, controls gene expression through regulation of the methylation status of cytosine residues in CpG dinucleotides of gene transcription regulation regions by DNA methyltransferase enzymes (12). DNA hypermethylation has been recognized as a common cause of gene silencing, and aberrant methylation can result in genomic instability which in turn may trigger cancer development (29, 36). Numerous studies have reported that DNA methylation plays important roles in hTERT transcription regulation (19). Loss of p16 expression through promoter hypermethylation is believed to be an essential step leading to tumor development (17). Furthermore, p16 activation by promoter hypomethylation mediates the senescence response in human cells (18). We therefore further investigated the mechanism of this induction through epigenetic pathways by examining the CpG methylation status of the hTERT and p16 promoters. We found that in normal WI-38 cells glucose reduction-induced DNA hypermethylation of the E2F-1 binding site, the active transcription factor of p16 (26, 27), may cause decreased binding of E2F-1 to the p16 promoter. This finding suggests that glucose restriction may induce p16 silencing by influencing DNA methylation processes and, therefore, affect binding of certain key transcription factors such as E2F-1 to the p16 promoter. This change in turn may contribute to lifespan extension in normal human cells. However, no methylation changes were found in the hTERT and p16 promoters of immortalized WI-38/S cells, indicating that DNA methylation may not be the main mechanism involved in the induction of gene expression by glucose restriction in precancerous cells.

To further understand the potential epigenetic mechanisms involving differential expression patterns of p16 and hTERT in normal and immortalized WI-38 cells induced by glucose restriction, we also assessed the regulation of p16 and hTERT expression by using two epigenetic modulators, 5-aza and TSA. We found that the promoter activities of p16 and hTERT can be more efficiently regulated by 5-aza and TSA in normal WI-38 cells, which is partly due to more prominent alterations of the DNMT and HDAC activities than that in immortalized WI-38/S cells in response to glucose restriction. These results may provide more clues underlining the phenomenon of differential gene expression patterns induced by energy stress in normal and immortalized cells, which will lead to various cellular fates such as apoptosis, senescence and cellular lifespan extension.

In summary, the findings from this study reveal epigenetic mechanisms by which glucose restriction may cause gene expression alteration in both normal and precancerous cells. More importantly, for the first time we found that glucose restriction-induced altered key gene expression through epigenetic mechanisms is a main pathway leading to different cell fates in normal and precancerous cells. Our study is consistent with the view that metabolic changes are associated with epigenetic changes in gene control during carcinogenesis and aging, and elucidating the complicated energy metabolism is beneficial to developing new approaches to cancer chemoprevention and therapy as well as antiaging approaches. Therefore, these findings have important implications for the application of energy control in cancer chemoprevention and therapy as well as extension of cellular longevity.

Acknowledgments

The authors thank Dr. Gordon Peters (Imperial Cancer Research Fund Laboratories, London, UK) and Dr. Silvia Bacchetti (Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada) for kindly providing the luciferase constructs used in this investigation.

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