Cloning and functional characterization of a novel gene, Romo1 = 새로운 유전자 Romo1의 클로닝 및 기능 규명

다국어 초록 (Multilingual Abstract)

Chemoresistance is one of the major limiting factors for the success of cancer chemotherapy. To clone a new gene related to drug resistance, differential display PCR was performed using tumor tissues from a patient who was initially sensitive to chemotherapy but became resistant to chemotherapy after recurrence. We cloned a novel gene and named it Romo1 (ROS modulator 1).
To investigate the biological function of the cloned gene, stable transfectant cells were made to observe the cytotoxicity toward antitumor agents (5-FU, cisplatin, paclitaxel, and doxorubicin) by cell counting. Because Romo1-overexpressing cells showed drug resistance to cell cycle specific drugs, flow cytometry analysis and Western blot analysis were carried out to investigate whether Romo1 was related to cell cycle regulation. To investigate the cellular localization of Romo1, a Romo1-GFP fusion protein was generated and its localization was observed under a fluoromicroscope. Cell cycle analysis by flow cytometry showed that Romo1-overexpressing cells were arrested at the G1 phase compared to control cells. Down-regulation of cyclin E and pRb was observed in Romo1-overexpressing cells. The Romo1-GFP fusion protein was specifically localized in mitochondria. Interestingly, increased production of ROS was observed in Romo1-overexpressing cells and its region was localized in mitochondria. To confirm the cellular function of Romo1, Western blot and FACS analysis were repeated after the treatment with an antisense oligomer of Romo1.
The results indicate that Romo1 enhances ROS generation and increases cell cycle arrest at G1. Also its function could be inhibited by treatment of antisense oligomers. In summary, Romo1 is a novel protein localized in mitochondria and enhances ROS generation, resulting in cell cycle arrest at G1