The study of differences between leukaemic cell line and normal cell in Wnt signaling pathway

• Main Authors: Jhih-Yi Teng, 滕芷儀
• Other Authors: Chien-Hui Lieu
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/83473726929655547920

碩士 === 國立陽明大學 === 醫學生物技術研究所 === 93 === Activation of Wnt/β-catenin signaling pathway in hematopoietic stem cell (HSC) appears to enhance the self-renewal activity and proliferation, and aberrant expression of this pathway involves in tumor progression. It is generally believed that leukemic stem cell (LSC) resembles HSC except that LSC escapes the normal proliferation regulation, which may relate to expression ofβ-catenin. The rationale of β- catenin overexpression in leukaemic cell remains unclear. We therefore investigated the difference between leukaemic cell line and normal cell in Wnt/β-catenin signaling pathway. Our data showed that leukaemic cell lines Jurkat, U937, K562, and HL-CZ were rhodamine 123 positive, indicating the non-HSC characters of these cells. Using immunofluorescence to detect β- catenin, we found that nucleus of all the leukemic cell lines stained positive, the peripheral monocytes stained weak positive in plasma, while lymphocytes stained negative. We then employed the real-time RT-PCR to assess the mRNA of four known β-catenin-related proteins. The mRNA expression for β-catenin , GSK3β, APC, and Axin were no difference in all cells detected. However, these four proteins could not be detected in normal peripheral mononuclear cell by Western blotting. Another study showed that β-catenin did not increased after 18 hours of incubation with PHA. This indicates that protein degradation must be a critical event for β-catenin overexpression. With cycloheximide blocking the protein synthesis, the β-catenin of U937 cell degraded rapidly within half hour, while that of K562, Jurkat and HL-CZ was sustained after 4 hours. Using immunoprecipitation method, we also found the β-catenin did not form complex with GSK3β, APC, or Axin in the Jurkat cell, which may lead to the β-catenin accumulation, and then enter the nucleus. Finally, the Porc gene, a Wnt protein activator, was detected in the leukemic cell line and also in blood of AML patients, but not in PBMNCs. In conclusion, our results showed that the differences between leukemia cell lines and PBMNC in Wnt signaling might be either the overexpression of Porc gene, defect in formation of β-catenin/GSK3β/ APC/Axin complex, or due to stimulation of Wnt proteins. The detail mechanism remains further study. In this study, we also found Jurkat is less mature than other leukemia cell lines and can be used as the model of studying the LSC.