Purpose: We have investigated whether the folate supplementation could be used to limit the aggressiveness of glioma through the DNA remethylation because (a) the cancer genome is characterized by a low level of DNA methylation (or 5-methylcytosine, 5 mC); and (b) folate is the main generator of S-adenosyl-methionine, the methyl donor molecule in the DNA methylation reaction catalyzed by the DNA methyltranferases. Experimental Design: The effects of folate supplementations were analyzed on the global DNA methylation status, the methylation status of DNA repeat element, the sensitivity of temozolomide-induced apoptosis, and the proliferation index of glioma cells. Finally, we analyzed whether the DNA methylation level could be used as a prognostic factor and/or a biomarker in an antiglioma therapy using folate supplementation as an adjuvant. Results: Our data show that gliomagenesis is accompanied by a reduction in 5 mC levels and that this low level of 5 mC is a poor prognostic factor in Glioblastoma Multiforme patients. We also show that folate supplementation enhanced the DNA remethylation through the Sp1/Sp3-mediated transcriptional up-regulation of genes coding for Dnmt3a and Dnmt3b proteins, two de novo methyltranferases. Finally, we show that the folate-induced DNA methylation limits proliferation and increases the sensitivity to temozolomide-induced apoptosis in glioma cells through methylation of the genes implicated in these processes (PDGF-B, MGMT, survivin, and bcl-w). Conclusion: This study suggests that folate supplementation could be a promising adjuvant for the future design of antiglioma therapies in preclinical and/or clinical studies.Despite the irrefutable role of genetic mechanisms in triggering tumorigenesis, epigenetic modifications, and particularly the DNA methylation modifications, are now recognized as frequent alterations playing a crucial role in the development and progression of human malignancies (1-3). Two distinct DNA methylation abnormalities are observed in cancer. The first is an overall genome-wide reduction in DNA methylation (global hypomethylation) and the second is regional hypomethylation or hypermethylation within the CpG islands of specific gene promoters. Both forms of hypomethylation are believed to induce proto-oncogene activation and chromosomal instability, whereas regional hypermethylation is strongly associated with transcriptional silencing of tumor suppressor genes (4). Thus, DNA methylation can function as a "switch" to activate or repress gene transcription, providing an important mechanism for overexpressed or silenced genes involved in the regulation of the cell cycle, DNA repair, growth signaling, angiogenesis, and apoptosis, and by ricochet in the initiation and the development of tumors (5).To date, an increasing number of reports investigating epigenetic signatures in malignant gliomas and more particularly the hypermethylation of tumor suppressor genes have been published. For example, it has been reported that PTEN methylation occurs frequen...