Methyl Donor Status Influences DNMT Expression and Global DNA Methylation in Cervical Cancer Cells.

BACKGROUND: Methyl donor status influences DNA stability and DNA methylation although little is known about effects on DNA methyltransferases. The aim of this study was to determine whether methyldonor status influences DNA methyltransferase (Dnmt) gene expression in cervical cancer cells, and if so, whether there are associated effects on global DNA methylation. MATERIALS AND METHODS: The human cervical cancer cell line, C4 II, was grown in complete medium and medium depleted of folate (FM+) and folate and methionine (FM). Growth rate, intracellular folate, intracellular methionine and homocysteine in the extracellular medium were measured to validate the cancer cell model of methyl donor depletion. Dnmt expression was measured by qRT PCR using relative quantification and global DNA methylation was measured using a flow cytometric method. RESULTS: Intracellular folate and methionine concentrations were significantly reduced after growth in depleted media. Growth rate was also reduced in response to methyl donor depletion. Extracellular homocysteine was raised compared with controls, indicating disturbance to the methyl cycle. Combined folate and methionine depletion led to a significant downregulation of Dnmt3a and Dnmt3b; this was associated with an 18% reduction in global DNA methylation compared with controls. Effects of folate and methionine depletion on Dnmt3a and 3b expression were reversed by transferring depleted cells to complete medium. CONCLUSIONS: Methyl donor status can evidently influence expression of Dnmts in cervical cancer cells, which is associated with DNA global hypomethylation. Effects on Dnmt expression are reversible, suggesting reversible modulating effects of dietary methyl donor intake on gene expression, which may be relevant for cancer progression.

Tumour suppressor gene methylation and cervical cell folate concentration are determinants of high-risk human papillomavirus persistence: a nested case control study.

BACKGROUND: Persistent infection with one or more high-risk human papillomavirus [HR-HPV] types increases the risk of intraepithelial neoplasia and cervical cancer. A nested case-control study was conducted to investigate the importance of cervical cell folate concentration and tumour suppressor gene methylation as risk factors for HR-HPV persistence. METHODS: Cervical cell samples from 955 women with HR-HPV infection and normal, borderline or mild dyskaryosis were retrieved from the archive of a population-based screening trial. Women were classified as cases or controls, reflecting the presence or absence [respectively] of any HR-HPV infection at a follow-up clinic at least 6 months from baseline. Cervical cell folate concentration and promoter methylation of five tumour suppressor genes were measured in independent samples from cases and controls. RESULTS: A higher cervical cell folate concentration [P = 0.015] was an independent predictor of infection at follow-up, together with infection with HPV-16 or infection with multiple HR-HPV types. Methylation of the tumour suppressor gene DAPK was associated with a 2.64-fold [95% CI, 1.35-5.17] increased likelihood of HPV infection whilst CDH1 methylation was associated with a 0.53-fold [95% CI, 0.331-0.844] likelihood of HR-HPV infection at follow-up. When considering women with normal or abnormal cytology, the predictive effect of higher cervical cell folate was only seen in women with mild cytology [P = 0.021]; similarly the effect of DAPK methylation was seen in women with mild or borderline cytology [P < 0.05]. CONCLUSIONS: Higher cervical cell folate concentration and promoter methylation of the tumour suppressor gene, DAPK, in women with cervical cell dyskaryosis, are associated with increased risk of HR-HPV persistence.

A vitamin B-12 supplement of 500 µg/d for eight weeks does not normalize urinary methylmalonic acid or other biomarkers of vitamin B-12 status in elderly people with moderately poor vitamin B-12 status.

Plasma vitamin B-12 is the most commonly used biomarker of vitamin B-12 status, but the predictive value for low vitamin B-12 status is poor. The urinary methylmalonic acid (uMMA) concentration has potential as a functional biomarker of vitamin B-12 status, but the response to supplemental vitamin B-12 is uncertain. A study was conducted to investigate the responsiveness of uMMA to supplemental vitamin B-12 in comparison with other biomarkers of vitamin B-12 status [plasma vitamin B-12, serum holotranscobalamin (holoTC), plasma MMA] in elderly people with moderately poor vitamin B-12 status. A double-blind, placebo-controlled, randomized 8-wk intervention study was carried out using vitamin B-12 supplements (500 µg/d, 100 µg/d, and 10 µg/d cyanocobalamin) in 100 elderly people with a combined plasma vitamin B-12 <250 pmol/L and uMMA ratio (µmol MMA/mmol creatinine) >1.5. All biomarkers had a dose response to supplemental vitamin B-12. Improvements in plasma vitamin B-12 and serum holoTC were achieved at cobalamin supplements of 10 µg/d, but even 500 µg/d for 8 wk did not normalize plasma vitamin B-12 in 8% and serum holoTC in 12% of people. The response in uMMA was comparable with plasma MMA; 15-25% of people still showed evidence of metabolic deficiency after 500 µg/d cobalamin for 8 wk. There was a differential response in urinary and plasma MMA according to smoking behavior; the response was enhanced in ex-smokers compared with never-smokers. uMMA offers an alternative marker of metabolic vitamin-B12 status, obviating the need for blood sampling.

Determinants of urinary methylmalonic acid concentration in an elderly population in the United Kingdom.

BACKGROUND: An age-related deterioration of vitamin B-12 status has been well documented. The early detection of deficiency may prevent the development of serious clinical symptoms, but plasma vitamin B-12 concentration is known to be an imperfect measure of vitamin B-12 status. Urinary methylmalonic acid (MMA) may be a more informative biomarker of vitamin B-12 status; however, biochemical, dietary, and other lifestyle determinants are not known. OBJECTIVE: We identified determinants of urinary MMA concentrations in free-living men and women aged ≥65 y in the United Kingdom. DESIGN: A cross-sectional study in 591 men and women aged 65-85 y, with no clinical evidence of vitamin B-12 deficiency, was conducted to determine the demographic, clinical, and lifestyle determinants of urinary MMA concentration expressed as the ratio of micromoles of MMA to millimoles of creatinine (uMMA ratio). RESULTS: Twenty percent of subjects had plasma vitamin B-12 concentrations <200 pmol/L. Seventeen percent of the variation in the uMMA ratio could be explained by plasma holotranscobalamin and sex; total vitamin B-12 intake and measures of renal function and gastric function made only a small contribution to the model. The uMMA ratio was lower in people with moderately impaired renal function. CONCLUSIONS: Plasma holotranscobalamin and sex were the most important determinants of uMMA ratio in elderly people with no clinical diagnosis of renal impairment. This biomarker might underestimate vitamin B-12 deficiency in a population in which renal impairment is prevalent. This trial was registered at www.controlled-trials.com as ISRCJN83921062.

Folate status and aberrant DNA methylation are associated with HPV infection and cervical pathogenesis.

Aberrant DNA methylation is a recognized feature of human cancers, and folate is directly involved in DNA methylation via one-carbon metabolism. Previous reports also suggest that folate status is associated with the natural history of human papillomavirus (HPV) infection. A cross-sectional study was conducted to test the hypothesis that folate status and aberrant DNA methylation show a progressive change across stages of cervical pathology from normal cells to cervical cancer. Additionally, we postulated that a gene-specific hypermethylation profile might be used as a predictive biomarker of cervical cancer risk. DNA hypermethylation of seven tumor suppressor genes, global DNA hypomethylation, systemic folate status, and HPV status were measured in 308 women with a diagnosis of normal cervix (n = 58), low-grade cervical intraepithelial neoplasia (CIN1; n = 68), high-grade cervical intraepithelial neoplasia (CIN2, n = 56; and CIN3, n = 76), or invasive cervical cancer (ICC; n = 50). Lower folate status was associated with high-risk HPV infection (P = 0.031) and with a diagnosis of cervical intraepithelial neoplasia or invasive cervical cancer (P < 0.05). Global DNA hypomethylation was greater in women with invasive cervical cancer than all other groups (P < 0.05). A cluster of three tumor suppressor genes, CDH1, DAPK, and HIC1, displayed a significantly increased frequency of promoter methylation with progressively more severe cervical neoplasia (P < 0.05). These findings are compatible with a role for folate in modulating the risk of cervical cancer, possibly through an influence over high-risk HPV infection. DAPK, CDH1, and HIC1 genes are potential biomarkers of cervical cancer risk.