Studies on abnormalities of membrane receptor signaling in malignant lymphoma cells as a basis of diagnosis and treatment

（1）CD 30启动子激活分析：CD 30组成性过表达激活ERK 1/2 MAPK通路，诱导JunB表达，这是肿瘤的共同特征。NF-kB的活化是由Hodgkin/Reed-斯滕贝格（H-RS）细胞上过表达的CD 30的“配体非依赖性活化”引起的，并且通过转导缺乏胞质区的显性负性诱饵CD 30来抑制CD 30信号传导诱导H-RS细胞的凋亡性细胞死亡（Horie et al.，Oncogene 21，2493，2002）。我们表明TRAF 2和TRAF 5蛋白质的细胞质聚集代表了CD 30的恒定信号传导（Horie et al.，Am J Pathol 160，1647，2002）。我们证明了位于微卫星附近的AP-1结合序列抵消了微卫星对CD 30启动子活性的抑制作用（Watanabe等人，Am J Pathol 163，633，2003）。我们发现p80 NPM-ALK通过过度表达抑制配体非依赖性信号转导。 ...更多信息 通过在NPM-ALK和野生型NPM的复合物中螯合TRAF蛋白，ALCL细胞中的CD 30（Horie等人，Cancer Cell 5，353，2004）。（2）原代ATL细胞的表达谱分析：利用合成的DNA芯片系统，对16例ATL患者和11例健康志愿者的原代ATL细胞的表达谱进行了分析。结果显示108个基因过度表达，250个基因下调。基于这些结果，我们现在正在制备使用多重实时PCR分析样品的PBMC中ATL细胞的检测系统。一项初步研究在来源于被认为是无症状携带者的携带者的样品中清楚地检测到ATL细胞，这在随后的Southern印迹分析中通过单克隆模式得到证实。（3）NF-kB靶基因的研究：应用CodeLink系统分析NF-kB组成型激活的细胞系和DHMEQ处理后的细胞表达谱。结果显示，超过1,000个基因被DHMEQ处理上调或下调了两倍以上。上调基因包括参与促凋亡功能的基因，下调基因包括参与抗凋亡功能的基因。此外，揭示了许多基因是NF-κ B信号传导的靶基因（这些数据的一部分用于BLOOD 106，2642，2005的手稿）。（4）DHMEQ抑制NF-kB分子靶向治疗的基础研究：通过体外实验和SCID小鼠体内移植瘤模型检测DHMEQ对ATL、CLL和霍奇金淋巴瘤的抗肿瘤活性。对于ATL，DHMEQ显示能够清除携带者PBMC中的HTLV-1感染的细胞，但不能清除转化的细胞（BLOOD 106，2642，2005）。DHMEQ对CLL细胞也有效，并且当组合使用时与氟达拉滨显示出协同效应（Leukemia 20，800，2006）。DHMEQ在体外和体内显示出对H-RS细胞的抗肿瘤活性（Cancer Res，提交）。少

(1)Analysis of CD30 promoter activation :We demonstrated that the constitutive overexpression CD30 activate ERK1/2 MAPK pathway, and induces JunB expression, which is a common characteristic of neoplastic. Activation of NF-kB results from "ligand-independent activation" of overexpressed CD30 on the Hodgkin/Reed- Sternberg (H-RS) cells, and that inhibition of CD30 signaling by transduction of a dominant negative decoy CD30 that lacks the cytoplasmic region induced apoptotic cell death of H-RS cells (Horie et al., Oncogene 21, 2493, 2002). We showed that cytoplasmic aggregation of TRAF2 and TRAF5 proteins represents constant signaling of CD30 ( Horie et al., Am J Pathol 160, 1647, 2002). We demonstrated that the AP-1 binding sequence located near the microsatellite counteracts the suppressive effects of the microsatellite on the CD30 promoter activity (Watanabe et al., Am J Pathol 163, 633, 2003). We showed that p80NPM-ALK inhibits ligand-independent signal transduction by overexpressed … More CD30 in ALCL cells though sequestration of TRAF proteins in the complex of NPM-ALK and wild type NPM (Horie et al., Cancer Cell 5, 353, 2004).(2)Expression profile analysis of primary ATL cells :We characterized the expression profile of primary ATL cells using the synthetic DNA array system and samples of 16 ATL cases and 11 healthy volunteers. The results revealed 108 overexpressed genes and 250 downregulated genes. Based on the results, we are now preparing a detection system of ATL cells in the PBMC using multiple real-time PCR analysis of samples. A pilot study clearly detected ATL cells in a sample derived from a carrier considered to be an asymptomatic carrier, which was confirmed by monoclonal pattern in subsequent Southern blot analysis.(3)Studies on the target genes of NF-kB :Expression profile analyses were done by CodeLink system using cell lines with constitutive activation of NF-kB and treatment by DHMEQ. The results revealed more than 1,000 genes that are up- or down-regulated more than two-fold by DHMEQ treatment. Up-regulated genes included these involved in proapoptotic function, and down-regulated genes included those involved in anti-apoptotic function. Furthermore, many genes were revealed to be target genes of NF-kB signaling (a part of these data were used in the manuscript of BLOOD 106, 2642, 2005).(4)Basic studies on the molecular targeted therapy by NF-kB inhibition using DHMEQ :Anti-tumor activity of DHMEQ against ATL, CLL and Hodgkin's lymphoma was tested by in vitro experiments and in vivo xenograft model using a SCID mice system. As to ATL, DHMEQ was shown to be able to purge carrier's PBMC of HTLV-1-infected but not transformed cells (BLOOD 106, 2642, 2005). DHMEQ was also effective against CLL cells, and showed synergistic effect with fludarabine when used in combination (Leukemia 20, 800, 2006). DHMEQ showed anti-tumor activity against H-RS cells in vitro and in vivo (Cancer Res, submitted). Less