Epigenetic Silencing of ALDH1L1, a Metabolic Regulator of Cellular Proliferation, in Cancers

FDH (10-formyltetrahydrofolate dehydrogenase, the product of the ALDH1L1 gene), a major folate-metabolizing enzyme in the cytosol, is involved in the regulation of cellular proliferation. We have previously demonstrated that FDH is strongly and ubiquitously down-regulated in malignant human tumors and cancer cell lines. Here, we report that promoter methylation is a major mechanism controlling FDH levels in human cancers. A computational analysis has identified an extensive CpG island in the ALDH1L1 promoter region. It contains 96 CpG pairs and covers the region between −525 and +918 bp of the ALDH1L1 gene including the promoter, the entire exon 1, and a part of intron 1 immediately downstream of the exon. Bisulfite sequencing analysis revealed extensive methylation of the island (76%-95% of CpGs) in cancer cell lines. In agreement with these findings, treatment of FDH-deficient A549 cells with the methyltransferase inhibitor 5-aza-2′-deoxycytidine restored FDH expression. Analysis of the samples from patients with lung adenocarcinomas demonstrated methylation of the ALDH1L1 CpG island in tumor samples and a total lack of methylation in respective normal tissues. The same phenomenon was observed in liver tissues: the CpG island was methylation free in DNA extracted from normal hepatocytes but was extensively methylated in a hepatocellular carcinoma. Levels of ALDH1L1 mRNA and protein correlated with the methylation status of the island, with tumor samples demonstrating down-regulation of expression or even complete silencing of the gene. Our studies have also revealed that exon 1 significantly increases transcriptional activity of ALDH1L1 promoter in a luciferase reporter assay. Interestingly, the exon is extensively methylated in samples with a strongly down-regulated or silenced ALDH1L1 gene

Activation of p21-Dependent G1/G2 Arrest in the Absence of DNA Damage as an Antiapoptotic Response to Metabolic Stress

The folate enzyme, FDH (10-formyltetrahydrofolate dehydrogenase, ALDH1L1), a metabolic regulator of proliferation, activates p53-dependent G1 arrest and apoptosis in A549 cells. In the present study, we have demonstrated that FDH-induced apoptosis is abrogated upon siRNA knockdown of the p53 downstream target PUMA. Conversely, siRNA knockdown of p21 eliminated FDH-dependent G1 arrest and resulted in an early apoptosis onset. The acceleration of FDH-dependent apoptosis was even more profound in another cell line, HCT116, in which the p21 gene was silenced through homologous recombination (p21−/− cells). In contrast to A549 cells, FDH caused G2 instead of G1 arrest in HCT116 p21+/+ cells; such an arrest was not seen in p21-deficient (HCT116 p21−/−) cells. In agreement with the cell cycle regulatory function of p21, its strong accumulation in nuclei was seen upon FDH expression. Interestingly, our study did not reveal DNA damage upon FDH elevation in either cell line, as judged by comet assay and the evaluation of histone H2AX phosphorylation. In both A549 and HCT116 cell lines, FDH induced a strong decrease in the intracellular ATP pool (2-fold and 30-fold, respectively), an indication of a decrease in de novo purine biosynthesis as we previously reported. The underlying mechanism for the drop in ATP was the strong decrease in intracellular 10-formyltetrahydrofolate, a substrate in two reactions of the de novo purine pathway. Overall, we have demonstrated that p21 can activate G1 or G2 arrest in the absence of DNA damage as a response to metabolite deprivation. In the case of FDH-related metabolic alterations, this response delays apoptosis but is not sufficient to prevent cell death

Prehabilitation in Cardiovascular Surgery: The Effect of Neuromuscular Electrical Stimulation (Randomized Clinical Trial)

Objective: We aimed to determine the effects of prehabilitation with neuromuscular electrical stimulation (NMES) on muscle status and exercise capacity in patients before cardiac surgery. Methods: Preoperative elective cardiac surgery patients were randomly assigned to the NMES group or control group. Intervention in the NMES group was 7–10 sessions, whereas the control group carried out breathing exercises and an educational program. The outcome measures included a six-minute walk test (6MWT) and a muscle status assessment (knee extensor strength (KES), knee flexor strength (KFS), and handgrip strength (HS)) after the course of prehabilitation. Results: A total of 122 patients (NMES, n = 62; control, n = 60) completed the study. During the NMES course, no complications occurred. After the course prehabilitation KES, KFS, and 6MWT distance were significantly increased (all p < 0.001) in the NMES group compared to the control. There was no significant difference in HS before surgery. Conclusions: A short-term NMES course before cardiac surgery is feasible, safe, and effective to improve preoperative functional capacity (six-minute walk distance) and the strength of stimulated muscles

Disposition of GNMT in cellular metabolism.

<p>GNMT converts SAM to SAH, methylating glycine to sarcosine. This reaction regulates SAM/SAH ratio and shuttles methyl groups, from activated methyl cycle back to the folate pool. Inhibitory effect of 5-CH3-THF (5-MTHF) on GNMT catalysis is indicated. Hcy, homocysteine; Sarc, sarcosine; THF, tetrahydrofolate.</p

Catalytically inactive or folate-binding deficient GNMT mutants are capable of the antiproliferative effect.

<p><b>A</b>. Crystal structure of GNMT tetramer (RCSB Protein Data Bank 3ths; subunits are shown in different colors) with bound 5-MTHF monoglutamate (two molecules shown in spacefill mode are bound per tetramer). <b>B</b>. Positions of amino acid residues in the GNMT catalytic center (from RCSB Protein Data Bank 1XVA). Acetate (Ac) is the competitive inhibitor of Gly and presumably occupies the same position in the active center. Glu 15 (E15*) is from a different subunit. Dotted lines indicate hydrogen bonds. <b>C</b> and <b>D</b>. The enzyme activities and CD spectra of GNMT mutants, analyzed in this study. <b>E</b>. The MTT proliferation assay of cells transfected with empty vector (control), wild type GNMT (WT), or corresponding mutants. <i>Error bars</i> represent ± S.D., <i>n =3</i>. <b>F</b>. Folate binding site at the GNMT subunit interface (as shown in panel <b>A</b>); Selected for mutagenesis are residues within close distance to 5-MTHF molecule (these residues are from all four subunits, which are denoted in parentheses). <b>G</b>. Binding of 5-MTHF by GNMT mutants. <i>Error bars</i> represent ± S.D., <i>n =2</i>. <b>H</b>. The MTT proliferation assay of cells transfected with empty vector (control), wild type GNMT (WT), or folate-binding deficient mutants mutants. <i>Error bars</i> represent ± S.D., <i>n =3</i>. <b>I</b>. The supplementation with excessively high media folate or SAM does not rescue cells from the GNMT antiproliferative effect. <i>Error bars</i> represent ± S.D., <i>n =3</i>.</p

Effect of GNMT transient transfection on cellular proliferation.

<p>Cell viability was assessed by MTT assay (absorbance at 570 nm reflects the number of live cells). <i>Error bars</i> represent ± S.D., <i>n =3</i>. Insets show levels of GNMT (Western blot) at different time points after transfection.</p

Subcellular localization-specific effects of GNMT.

<p><b>A</b>. Sequences used to target GNMT to cytosol or nucleus. B. Distribution of GNMT fusion constructs between cytosol and the nucleus. All constructs included GFP tag at the C-terminus of GNMT. Respective subcellular targeting sequences were introduced at the C-terminus of the GFP tag. <b>C</b>. Levels of corresponding GNMT constructs after transient transfection (Western blot assay). <b>D</b>. MTT assay of cells transfected with GNMT constructs. <i>Error bars</i> represent ± S.D., <i>n =2</i>; *, <i>p < 0</i>.<i>05</i>.</p

Cellular responses to GNMT expression.

<p>A. Distribution of GNMT-expressing cells (<i>right panel</i>) between cell cycle phases (propidium iodide staining) compared to control (<i>left panel</i>) GNMT-deficient cells. <b>B</b>. Assessment of DNA damage in GNMT expressing cells by the Comet assay. <b>C</b>. Apoptotic cells assessed by Annexin V/propidium iodide staining after GNMT expression (<i>bottom right quadrant</i>, early apoptotic cells; <i>upper right quadrant</i>, late apoptotic cells); only green cells (expressing GFP-GNMT) were evaluated. <b>D</b>. Calculation of apoptotic cells from C. <b>E</b>. Activation of ERK phosphorylation in response to GNMT expression. <b>F</b>. zVAD-fmk, but not ERK inhibitor PD98059, partially rescues cells from the antiproliferative effect of GNMT (data for A549 cells are shown).</p