Search for the regulatory factor of apoptosis in adult T-cell leukemia (ATL) cells

1. 抗fas单克隆抗体（mAb）耐药的成人t细胞白血病（ATL）细胞株RS04RS04 RS04细胞经IgM抗fas单抗处理后未出现凋亡。我们发现，作为蛋白体特异性抑制剂的lactacystin在RS04细胞中激活caspase 8，诱导细胞凋亡，说明RS04细胞胞质中凋亡通路是完整的。通过对Fas基因的测序分析，我们发现了一个杂合错义突变，在第2外显子A向G过渡，导致精氨酸取代组氨酸。突变Fas蛋白的三维分子模型表明，氨基酸52至58周围的构象发生了明确的改变。提示Fas分子的三聚化干扰突变的Fas，以显性负性机制导致凋亡信号转导受损。抗fas mAb抗atl细胞系OMTIn - OMT细胞，我们发现在核苷酸1132处错义点突变，T向C转变，导致苏氨酸取代异亮氨酸。这种突变可能导致死亡结构域的构象改变，不能与Fas分子正下游的FADD分子结合。由于Fas基因突变在Fas耐药的ATL细胞系中检测到的比例相对较高，因此我们研究了几乎不表达膜Fas且对IgM抗Fas单抗耐药的ATL细胞。在7例ATL患者中，我们发现4例Fas基因突变，3个点突变和2个碱基对缺失。提示Fas基因的多种突变是阻止ATL细胞凋亡的机制之一。

1. Anti-Fas monoclonal antibody (mAb) resistant adult T-cell leukemia (ATL) cell line, RS04RS04 RS04 cells did not show apoptosis after treatment with IgM anti-Fas mAb. We found that the lactacystin, which was a specific inhibitor of the proteosome, activated the caspase 8 and induced apoptosis in RS04 cells, indicating that the apoptotic pathway in the cytoplasm in RS04 cells was intact. By sequencing analysis of Fas gene, we found the heterozygous missense mutation with the transition of A to G at exon 2, resulting in substitution of arginine for histidine. The three dimensional molecular modeling of the mutated Fas protein indicated the definitive conformational alteration around the amino acids 52 to 58. It was suggested that the trimerization of Fas molecules was interfered with the mutated Fas, resulting in the impairment of signal transduction of apoptosis by dominant negative mechanism.2. Anti-Fas mAb resistantATL cell line, OMTIn OMT cells, we found the missense point mutation at nucleotide 1132 with the transition of T to C, resulting in the substitution of threonine for isoleucine. This mutation might cause the conformational alteration of death domain and fail to associate with the FADD molecule, which located at right downstream under the Fas molecule.3. ATL cells which barely express membrane FasBecause the Fas gene mutations were detected at the relatively high rate in Fas-resistant ATL cell lines, we investigated the primary ATL cells, which barely expressed membrane Fas and were resistant to IgM anti-Fas mAb. In four of seven cases of patients with ATL, we found Fas gene mutations, three point mutations and a 2 base pair deletion. It was indicated that the various mutations of Fas gene were one of the mechanisms of preventing the apoptosis in ATL cells.