Ras 암 유전자에 의한 세포 생존연구

Abstract

Point mutations in the mammalian ras gene are oncogenic, and occur in approximately 30 % of human tumors. Although several studies concerning the role of oncogenic H-ras in the expression of multidrug resistance (MDR) in various cell lines, the role of ras in the regulation of MDR need to be elucidated. Therefore, we investigate the role of oncogenic H-ras in rat mdr1b expression of NIH3T3 cells. The constitutive expression of oncogenic H-ras (V12-ras) was found to down-regulate mdr1b promoter activity and mdr1b mRNA expression. The mdr1b promoter activity of V12-ras-NIH3T3 cells was 75 % less than pcDNA3-NIH3T3 cells and the level of mdr1b mRNA expression in pcDNA3-NIH3T3 cells were approximately 3-fold increased than that of V12-ras-NIH3T3 cells. In addition, treatment of pcDNA3-NIH3T3 cells with 50, 100 and 250 nM of doxorubicin led to increase mdr1b-Luc activity by 150-, 200- and 400-fold compared with untreated cells, respectively. However, in V12-ras-NIH3T3 cells, the corresponding enhancements of mdr1b-Luc activities were only 100-, 120-, and 150-fold versus untreated cells. Moreover, there is a strong correlation between the mdr1b expression and a sensitive to doxorubicin toxicity. To examine the detailed mechanism of V12-ras-mediating down-regulation of mdr1b expression, antioxidant NAC and the NADPH oxidase inhibitor DPI were used. Pretreatment of cells with either NAC or DPI was found to significantly enhance oncogenic H-ras-mediated down-regulation of mdr1b expression and to markedly prevent doxorubicin-induced cell death. As extracellular signal-regulated kinase (ERK) is a known downstream effector of Ras and to be stimulated by ROS, we next investigated whether alterations in ERK signaling affect mdr1b expression. NAC and DPI treatment led to decrease the ERK activity, and the ERK inhibitors PD98059 or U0126, caused enhancement of the mdr1b-Luc activity of V12ras-NIH3T3 and reduced doxorubicin-induce apoptosis. Taken together, these results suggest that oncogenic H-ras expression could down-regulate the mdr1b expression through intracellular ROS production, and the activation of ERK induced by ROS, at least in part, involved in the down-regulation of mdr1b expression in NIH3T3 cells.|Ras의 점돌연변이는 인간 암세포 전체의 약 30%에서 발견된다. 돌연변이를 통한 활성화된 Ras 종양유전자는 정상세포를 형질전환 할 수 있고 악성 사람 종양들의 많은 개체의 성장에 관련되어있다. Ras 암 유전자 가 활성화된 암세포는 암세포의 생존률을 조절하는데 아직 그 기작이 밝혀지지않았다. 본 연구 논문에서는 활성화된 Ras 암유전자에 의한 암세포 생존증가가 어떠한 기작에 의하여 발생되는지를 규명하고자 하였다. 그 결과 본 연구 논문에서 는 항암제 내성을 유발하는 mdr1의 발현이 Ras 암유전자에 의하여 발현이 감소되었다. 또한 H-RasV12-transformed NIH3T3 세포들은 대조세포에비하여 mdr1 프로모터 활성 (mdr1-luc)이 현저하게 감소되었다. Ras 암 유전자에의한 mdr1 발현 감소기작을 규명하기 위하여 활성산소 억제제를 투여한 결과 Ras 암유전자에의하여 활성이 감소된 mdr1 프로모터는 활성산소 억제제인 NCA 또는 DPI투여시 정상화 되는것을 관찰되었으며 항암제인 doxorubicin에의한 암세포 생존에 영향을 미침을 규명하였다. 또한 ERK 억제제인 PD98059와 U0126을 투여 시 Mdr1의 프로모터 활성이 정상화 되었다. 이러한 결과들로 Ras 암유전자는 Ras downstrem 신호전달경로인 ERK활성을 증가시켜 활성산소를 발생시키며 이러한 활성산소가 mdr1의 발현을 조절하여 암세포 생존에 영향을 줄것으로 판단되었다.