|Budget Amount *help
¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 1998 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1997 : ¥2,300,000 (Direct Cost : ¥2,300,000)
Adult T-cell leukemia (ATL) is an aggressive neoplasm of helper T-lymphocytes, which is etiologically associated with human T-cell leukemia virus type I (HTLV-I). Siince its long latent period from infection of HTLV-I to onset of ATL, multti-step mechanism of leukemogenesis is considered in ATL.We are trying to identify the genes responsible for the onset and the progression of ATL.Although leukemic cells in most ATL cases expressed Fas antigen, we found Fas-negative cases. Neutrophil from this patient had Fas antigen on the surface, showing that Fas-negative phenotype is specific to leukemie cells. Sequences of RT-PCR products revealed that skipping of exon 4, and small deletion (5bp) caused premature termination of Fas protein synthesis, resulting in Fas negative phenotype. This Fas negative ATL cells were resistant to doxorubicin -induced apoptosis in vitro. Fas negative phenotype is considered to be associated with drug resistance.
Methylation of the p16^<INK4A> gene has been recogn
ized as another mechanism, in addition to somatic DNA changes such as deletion or mutation, which can inactivate the pl6^<INK4A> gene in various malignancies including melanoma, bladder cancer and malignant lymphoma. We analyzed the methylation of the p16^<INK4A> gene in patients with different subtypes of adult T-cell leukemia (ATL). Using Southern blot analysis and methylation-specific PCR (MSPCR), we detected methylation of the p16^<INK4A> gene was more frequently in acute ATL (49%) or lymphoma-type ATL (73%) than in lowgrade malignant stage, chronic (17%) and smoldering types (17%). In contrast, no methylation of the pl6^<INK4A> gene was found in asymptomatic HTLV-I carriers and uninfected control, and methylation of the p15 gene, which encode another inhibitor of CDK4 and 6, could not be found in any ATL samples : methylation of the p16^<INK4A> gene is highly specific for ATL.Deletion of the the p16^<INK4A> gene was found in another 20% of acute ATL patients by Southern blot analysis, and therefore, abnormalities of the p16^<INK4A> gene in acute ATL total to 69%. Furthermore, direct sequencing of the pl6^<INK4A> gene after sodium bisulfite treatment of genomic DNA revealed that the methylation of CpG sites occurred in most (23 out of 29) of ATL cases including chronic or smoldering ATL, even in the cases which methylation could not be detected by MSPCR or the Southern blot method. Semi-quantitative PCR showed markedly decreased p16^<INK4A> mRNA levels in the samples having a methylated p16^<INK4A> gene. These findings show that a level of CpG methylation plays an important role in the progression of ATL by further decreasing the expression of p16.
3) p16遺伝子は癌抑制遺伝子であるが様々な癌での欠失、変異が報告されている。一方、メチル化などのepigeneticな変化もp16遺伝子の抑制を起こす。methylation-specific PCR(MSPCR)による解析でATL細胞での高率なメチル化の存在が示された。さらにsodium bisulfite処理DNAを用いてPCRを行いdirect sequenceによって塩基配列を決定することによってメチル化を検討した。p16遺伝子は成人T細胞白血病細胞で高率に(86%)メチル化を受けていることを見出した。またメチル化の程度は臨床的に悪性度が増すに従い増強し、それに伴い転写産物量も減少した。脱メチル化を起こす5-aza-2′-deoxycytidineによってp16遺伝子転写は再活性化され確かにメチル化が転写抑制に関わっていることが明かとなった。 Less