ATM Radio-Gene Therapy for Brain Tumor

• Main Authors: Chu-Chiao Wu, 吳竹喬
• Other Authors: Chi-Shiun Chiang
• Format: Others
• Language: en_US
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/36807681526018717826

碩士 === 國立清華大學 === 生醫工程與環境科學系 === 94 === ATM (Ataxia telangiectasia-muted) gene plays an important role in the control of cell cycle, signal transduction, DNA repair and cell death after cell damage by irradiation. The involving functions and the range are magnified by the downstreams of ATM gene. However, the mechanism and role of ATM in the radioresistance of brain cancer remained largely unclear. In studies of gene therapy, TFO (triplex forming oligonucleotide) system and RNAi (RNA interference) system are believed to have functions of gene inhibition in DNA and RNA level, respectively. However, there were no published data to compare their inhibition effects. This study aimed to compare the ATM inhibition by these two systems and investigate the role of ATM played in the response of glioma to radiation therapy. To study the function of TFO, three TFOs that target different sequences of ATM gene were designed and assayed. The results showed that three TFOs could bind to their duplex targets. The melting point of the triplex is greater than 37℃ which indicates the possibility of triplex formation within cells under normal physiological condition. Resuls also showed that TFO could be efficiently delivered into cells. Delivery efficiency assay indicated that TFO were found in almost 90％ cells 12 hr after TFO delivery. Gene inhibition time course analysis found that the maximum inhibition effects on mRNA and protein levels occurred at 24 hr and 48- 52 hr after TFO transfection, respectively. Besides, three TFOs had different time-course of inhibitions because of their different composition. Among relevant research, this is the first experiment using TFO system to inhibit ATM gene expression. Regarding the construction of RNAi system, four virus vectors containing shRNAs for ATM inhibition were constructed, purified, and assayed. All of four shRNA expression cassettes had significant inhibition effects on mRNA levels at 12 hr after transfection. Corresponding stable-inhibition cell lines were constructed and specifically inhibited ATM expression was determined. We have also found that ATM stable-inhibition cell line had a retarded growth and different colony morphology. Moreover, the ROS increased in cells with inhibited ATM. Finally, the survival of U87MG glioma with suppressed ATM after irradiation was determined. The lack of ATM expression led to increase radiosensitivity in glioma U87MG but not in cervical carcinoma Hela. Besides, ATM inhibition influenced SLDR and PLDR ability of U87MG. The above results suggested that ATM inhibition may enhance radiation killing on glioma cells (U87MG) by affecting its SLDR and PLDR ability, and be associated with antioxidant function. However, further in vivo experiments were required to better define the role of ATM in human brain cancer to radiation therapy.