The influence of methyl donor status on cervical cancer cell behaviour in vitro

Epidemiological studies suggest that the availability of methyl-donor nutrients may influence the risk of cervical cancer and of cancer progression. This effect may be mediated by altered DNA instability and aberrant DNA methylation. The aim of this study was to develop and validate an appropriate in vitro model of methyl donor depleted cervical cancer cells and to use this model to investigate effects of methyl donor depletion on cell processes relevant to cancer risk and progression. The human cervical cancer cell lines, C4-II and SiHa were selected for study. Cells were grown in complete medium and in medium depleted of folate alone and in medium depleted of methionine and folate. Growth rate, intracellular folate, intracellular methionine, and extracellular homocysteine were measured to validate the cervical cancer cell model of methyl donor depletion. The depleted cells showed >20-fold reduction of intracellular folate concentration in both cell models and a 60% reduction of methionine concentration in C4-II cells. Methyl donor depletion led to a greater reduction in the growth rate of C4-II than SiHa cells. Extracellular homocysteine was significantly raised by methyl donor depletion compared with controls. Global DNA methylation was measured using a flow cytometric method. Combined depletion of folate and methionine led to an 18% reduction in global DNA methylation in C4-II cells but had no significant effect in SiHa cells; accordingly, only C4-II cells were used for further study. The expression of DNA methyl transferases (DNMTs) was measured using semi quantitative RT-PCR. Methyl donor depletion led to a significant down-regulation of DNMT3a and DNMT3b, which showed a 2.62 (P= 0.024) and 3.60-fold (P= 0.001) reduction in expression respectively. Total DNMT activity was increased by folate and methionine depletion cells (P= 0.001), although there are concerns about the quality of the assay used. Effects of folate and methionine depletion on cell growth, and DNMT3a and DNMT3b expression were reversed by transferring depleted cells to growth in complete medium. Gene microarray analysis was carried out in order to identify genes and pathways affected by methyl donor depletion. A high proportion of genes associated with cell death, cell communication, and cell motion were up-regulated whilst a high proportion of genes associated with regulation of the cell cycle, cytoskeleton organisation, and chromosome organisation were down-regulated. Despite findings from the gene microarray were suggestive of effects on cell migration rate this was not found to be significantly influenced by methyl donor depletion, using the scratch assay. Methyl donor status reversibly influences DNMT expression in cervical cancer cells in vitro, which may explain effects on global hypomethylation. Alterations in expression of other genes in response to methyl donor depletion are suggestive of cancer-promoting effects.