Biochemical analysis of tumor rejection antigen and its role of oncogenesis.

RL*1特异性CTL识别的抗原肽通过反相高效液相色谱分离部分的直接测序确定。分子分析表明，该抗原肽来自原癌基因c-AKT的5‘非转录区，通过插入一个小鼠白血病病毒LTR和6个未知来源的核苷酸而获得。在这项研究中，我们研究了这种改变的Akt分子及其在RL*1.1中的致癌作用。用兔抗c-Akt人工合成多肽抗体进行Western印迹分析，发现RL*1细胞除含有56kD的正常c-Akt蛋白外，还含有59kD的蛋白。2.59 kD突变Akt分子(RL-Akt)的表达水平约为正常c-Akt的10倍，在细胞膜部分和胞浆部分均有表达，而c-Akt仅在细胞质部分表达。3.RL-AKT转基因NIH3T3细胞生长良好，在软APR中形成集落。4.建立了RL-Akt缺失突变体和RL-Akt弱表达突变体。5.为了评价RL-Akt对BALB/c小鼠体内生长速度的影响，将这些突变体和亲本RL*1细胞分别移植到BALB/c小鼠体内，亲本RL*1细胞生长迅速，表达弱的突变体生长缓慢，缺失突变体生长更慢，提示RL-Akt在细胞膜上的过度表达和定位导致了生长因子-PI3途径的有效信号转导，参与了RL*1白血病的恶性转化。有关RL-Akt对宿主免疫系统影响的研究正在进行中。

The antigen peptides recognized by RL*1 specific CTL were determined by direct sequencing of the fraction separated by reversed-phase high performance liquid chromatography. Molecular analysis revealed the antigen peptide were derived from the 5'-untransrated region of proto-oncogene c-akt by insertion of a murine leukemia virus LTR together with six nucleotides of unknown origin. In this study, we investigated this altered Akt molecule and its role of oncogenesis in RL*1.1. Western blot analysis using plyclonal rabbit anti c-Akt synthetic polypeptide antibodies revealed a protein 59 KD in addition to 56 KD normal c-Akt protein in RL*1 cells. 2. The 59 KD altered Akt molecule (RL-Akt) was expressed at about ten times level to normal c-Akt and was detected in the membrane fraction and in the cytosolic fraction, although the c-Akt was detected only in the cytosolic fraction. 3. RL-akt transgeneic NIH 3T3 cells grew well, forming colonies in soft apr. 4. RL-Akt deletion mutant and RL-Akt weakly expressed mutant were established. 5. To assess the effect of RL-Akt in the growth rate in vivo, these mutants and parental RL*1 cells transplanted in BALB/c mice, respectively, parental RL*1 cells grow rapidlly, but weakly expressed mutant grow slowly, and deletion mutant did more slowly.These observations suggests that the over expression and localization in the membrane of RL-Akt resulted in effective signal transduction of growth factor-PI3 pathway and these participates in malignancy in RL*1 leukemia. The study of the effects of RL-Akt to the host immune system are going now.