Tatsunori Sakai

Identification of aberrantly methylated genes in association with adult T-cell leukemia

In this study, we identified 53 aberrantly hypermethylated DNA sequences in adult T-cell leukemia (ATL) cells using methylated CpG island amplification/representational difference analysis method. We also observed a proportionate increase in the methylation density of these regions 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 might be associated with DNA hypermethylation. To study their functions in ATL cells, we transfected recombinant adenovirus vectors expressing KLF4 and EGR3 genes. Expression of KLF4 induced apoptosis of ATL cells whereas enforced expression of EGR3 induced the expression of FasL gene, resulting in apoptosis. Thus, suppressed expression of EGR3 enabled ATL cells to escape from activation-induced cell death mediated by FasL. Our results showed that the methylated CpG island amplification/representational difference analysis method allowed the isolation of hypermethylated DNA regions specific to leukemic cells and thus shed light on the roles of DNA methylation in leukemogenesis.

A potential link between alternative splicing of the NBS1 gene and DNA damage/environmental stress.

Abstract Takai, K., Sakamoto, S., Sakai, T., Yasunaga, J., Komatsu, K. and Matsuoka, M. A Potential Link between Alternative Splicing of the NBS1 Gene and DNA Damage/Environmental Stress. Radiat. Res. 170, 33–40 (2008). NBS1 forms a multimetric complex with MRE11/RAD50, which acts as the sensor of DNA double-strand breaks (DSBs). The mechanisms controlling the expression of NBS1 remain largely unknown. Here we show that NBS1 is transcribed as both a wild-type and an alternatively spliced form exhibiting a premature stop codon in an alternative 50-bp exon in intron 2. Although the wild-type transcript predominates in most tissues, the spliced transcript is abundant in resting peripheral blood mononuclear cells (PBMCs). Levels of the spliced form of NBS1 decreased rapidly after irradiation as levels of the wild-type NBS1 transcript increased, resulting in increased levels of NBS1 protein. Both mitogenic stimulation and methyl methanesulfonate treatment also altered the splicing pattern of NBS1. Resting PBMCs, which predominantly express spliced NBS1, were more susceptible to radiation than mitogen-stimulated cells, which showed predominant expression of the wild-type transcript. Since the alternatively spliced NBS1 gene likely did not produce protein, this alternative splicing seems to be associated with the control of NBS1 protein. Thus alternative splicing of the NBS1 gene may be associated with the regulation of NBS1 in response to DSBs, DNA alkylation damage, and mitogenic response.

Elderly ATL patients in ageing society of Japan

Results More than 70% of newly diagnosed ATL patients were elderly persons aged 65 or older. Twenty-three of 66 patients at age from 41 to 61-year-old underwent allogeneic hematopoietic stem cell transplantation (alloHSCT) in our hospital. On the other hand, 38 patients were not suitable for allo-HSCT therapy. Their ages were from 51 to 89-year-old and elderly aged 65-yearold and over occupied 73.3% of them (22 of 30, who we confirmed their outcome). Seven patients of them died before treatment or during the primary therapy. Then, we focused patients cause acute transformation from chronic type to figure out the timing of primary intervention and the efficiency of the therapy. Elevating sCD30 was observed earlier than other markers (sIL-2R, LDH) due to acute transformation and reduced by the treatment regimen changed even during treatment of relapse (recurrence).