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

(1) CD30启动子激活分析：我们发现组成型过表达CD30激活ERK1/2 MAPK通路，诱导JunB表达，这是肿瘤的共同特征。NF-kB的激活源于霍奇金/里德-斯特恩伯格（H-RS）细胞上过表达的CD30的“不依赖配体的激活”，而CD30信号的抑制通过缺乏细胞质区的显性阴性诱饵CD30的转导诱导H-RS细胞的凋亡细胞死亡（Horie等人，Oncogene 21, 2493, 2002）。我们发现TRAF2和TRAF5蛋白的细胞质聚集代表CD30的恒定信号传导（Horie et al., Am J Pathol 160,1647,2002）。我们证明位于微卫星附近的AP-1结合序列抵消了微卫星对CD30启动子活性的抑制作用（Watanabe et al., Am J Pathol 163, 633, 2003）。我们发现p80NPM-ALK通过隔离NPM- alk和野生型NPM复合物中的trf蛋白，在ALCL细胞中过表达更多CD30来抑制配体非依赖性信号转导（Horie et al., Cancer Cell 5, 353, 2004）。(2) ATL原代细胞表达谱分析：采用合成DNA阵列系统，对16例ATL病例和11名健康志愿者的ATL原代细胞表达谱进行了表征。结果显示108个基因过表达，250个基因下调。基于这些结果，我们正在准备一套PBMC中ATL细胞的检测系统，对样品进行多重实时PCR分析。一项初步研究清楚地在一名被认为是无症状携带者的携带者的样本中检测到ATL细胞，并在随后的Southern blot分析中通过单克隆模式证实了这一点。(3) NF-kB靶基因的研究：利用CodeLink系统分析NF-kB组成激活细胞系和DHMEQ处理后的表达谱。结果显示，通过DHMEQ治疗，超过1000个基因的上调或下调幅度超过两倍。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，许多基因被发现是NF-kB信号的靶基因（这些数据的一部分被用于BLOOD 106, 2642, 2005的手稿中）。(4) DHMEQ抑制NF-kB分子靶向治疗的基础研究：采用SCID小鼠系统体外实验和体内异种移植模型检测DHMEQ对ATL、CLL和霍奇金淋巴瘤的抗肿瘤活性。对于ATL， DHMEQ被证明能够清除载体PBMC中htlv -1感染的细胞，但不能清除转化细胞（BLOOD 106, 2642, 2005）。DHMEQ对CLL细胞也有效，并与氟达拉滨联合使用时显示协同效应（白血病20,800,2006）。DHMEQ在体外和体内均显示出对H-RS细胞的抗肿瘤活性（Cancer Res, submitted）。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