Epigenetic modification in the nuclear genome plays a key role in human tumorigenesis. In this paper, we investigated whether changes in the mtDNA copy number frequently reported to vary in a number of human tumors induce methylation changes in the nucleus. We utilized the Restriction Landmark Genomic Scanning (RLGS) to identify genes that undergo changes in their methylation status in response to the depletion and repletion of mtDNA. Our study demonstrates that depletion of mtDNA results in significant changes in methylation pattern of a number of genes. Furthermore, our study suggests that methylation changes are reversed by the restoration of mtDNA in cells otherwise lacking the entire mitochondrial genome. These studies provide the first direct evidence that mitochondria regulate epigenetic modification in the nucleus that may contribute to tumorigenesis.

BackgroundFolate (vitamin B9) is essential for cellular proliferation as it is involved in the biosynthesis of deoxythymidine monophosphate (dTMP) and s-adenosylmethionine (AdoMet). The link between folate depletion and the genesis and progression of cancers of epithelial origin is of high clinical relevance, but still unclear. We recently demonstrated that sensitivity to low folate availability is affected by the rate of polyamine biosynthesis, which is prominent in prostate cells. We, therefore, hypothesized that prostate cells might be highly susceptible to genetic, epigenetic and phenotypic changes consequent to folate restriction.ResultsWe studied the consequences of long-term, mild folate depletion in a model comprised of three syngenic cell lines derived from the transgenic adenoma of the mouse prostate (TRAMP) model, recapitulating different stages of prostate cancer; benign, transformed and metastatic. High-performance liquid chromatography analysis demonstrated that mild folate depletion (100 nM) sufficed to induce imbalance in both the nucleotide and AdoMet pools in all prostate cell lines. Random oligonucleotide-primed synthesis (ROPS) revealed a significant increase in uracil misincorporation and DNA single strand breaks, while spectral karyotype analysis (SKY) identified five novel chromosomal rearrangements in cells grown with mild folate depletion. Using global approaches, we identified an increase in CpG island and histone methylation upon folate depletion despite unchanged levels of total 5-methylcytosine, indicating a broad effect of folate depletion on epigenetic regulation. These genomic changes coincided with phenotype changes in the prostate cells including increased anchorage-independent growth and reduced sensitivity to folate depletion.ConclusionsThis study demonstrates that prostate cells are highly susceptible to genetic and epigenetic changes consequent to mild folate depletion as compared to cells grown with supraphysiological amounts of folate (2 μM) routinely used in tissue culture. In addition, we elucidate for the first time the contribution of these aspects to consequent phenotype changes in epithelial cells. These results provide a strong rationale for studying the effects of folate manipulation on the prostate in vivo, where cells might be more sensitive to changes in folate status resulting from folate supplementation or antifolate therapeutic approaches.

Folate (vitamin B9) is utilized for synthesis of both S-adenosylmethionine (AdoMet) and deoxythymidine monophosphate (dTMP), which are required for methylation reactions and DNA synthesis, respectively. Folate depletion leads to an imbalance in both AdoMet and nucleotide pools, causing epigenetic and genetic damage capable of initiating tumorigenesis. Polyamine biosynthesis also utilizes AdoMet, but polyamine pools are not reduced under a regimen of folate depletion. We hypothesized that high polyamine biosynthesis, due to the high demand on AdoMet pools, might be a factor in determining sensitivity to folate depletion. We found a significant correlation (P<0.001) between polyamine biosynthesis and the amount of folate required to sustain cell line proliferation. We manipulated polyamine biosynthesis by genetic and pharmacological intervention and mechanistically demonstrated that we could thereby alter AdoMet pools and increase or decrease demand on folate availability needed to sustain cellular proliferation. Furthermore, growing a panel of cell lines with 100 nM folate led to imbalanced nucleotide and AdoMet pools only in cells with endogenously high polyamine biosynthesis. These data demonstrate that polyamine biosynthesis is a critical factor in determining sensitivity to folate depletion and may be particularly important in the prostate, where biosynthesis of polyamines is characteristically high due to its secretory function.