Aberrantly Hypermethylated Genes in Adult T-Cell Leukemia Cells: The Implications in the Leukemogenesis.

Abstract

Adult T-cell leukemia (ATL) is a highly aggressive neoplasm of helper T lymphocytes and is etiologically associated with human T-cell leukemia virus type I (HTLV-I). Although HTLV-I encoded Tax protein is thought to play a central role in the leukemogenesis of ATL, ATL cells frequently could not produce Tax protein by the somatic mutations, deletion and loss of 5′-LTR. Moreover, a long-term latent period (almost 60 years in Japan) precedes the onset of ATL, suggesting that multistep tumorigenesis is involved in development of ATL in addition to role of viral protein. Such transformation process is thought to include alterations of host genome: genetic and epigenetic changes. The epigenetic alteration, such as DNA methylation and histone modification, is commonly observed in various cancer cells. Recently, we reported that the aberrant expression of MEL1S gene, which is hypomethylated in ATL cells, confers resistance against transforming growth factor-beta.In this study, we identified 53 aberrantly hypermethylated DNA sequences in ATL cells using methylated CpG island amplification/representational difference analysis (MCA/RDA) method. In addition, we found that the DNA methylation of these regions tends to accumulate with disease progression. Seven genes, which were expressed in normal T-cells, but suppressed in ATL cells, were identified near the hypermethylated regions. Among these silenced genes, Kruppel-like factor 4 (KLF4) gene is a cell cycle regulator and early growth response 3 (EGR3) gene is a critical transcriptional factor for induction of Fas ligand (FasL) expression. Treatment with 5-aza-2′-deoxycytidine resulted in the recovery of their transcription, indicating that their silencing is associated with DNA hypermethylation. To study their role in oncogenesis of ATL, we transfected recombinant adenovirus vectors expressing KLF4 and EGR3 genes into ATL cell lines. Expression of KLF4 induced apoptosis of ATL cells whereas enforced expression of EGR3 induced the expression of FasL gene, resulting in apoptosis. This EGR3-induced apoptosis has been inhibited by stable transfection of cellular and viral FLIP, which suppress the activation of caspase-8 by preventing pro-caspase-8 in Fas-mediated apoptosis pathway. Therefore, suppressed expression of EGR3 enabled ATL cells to escape from activation-induced cell death mediated by Fas-FasL signaling. Our results showed that the DNA hypermethylation plays an important role in the leukemogenesis of ATL by silencing transcriptions of genes that inhibit the abnormal growth of ATL cells.