암/정소 항원 CAGE의 세포주기 촉진에 관한 기전연구 = The CAGE Cancer/Testis Antigen Promotes Cell Cycle Progression by Up-regulating Expression of Cyclin D1 and Cyclin E in AP-1 and E2F-1-dependent Manner

다국어 초록 (Multilingual Abstract)

A cancer/testis antigen, CAGE was shown to be widely expressed in various cancer tissues and cancer cell lines, but not in normal tissues except the testis. To examine the functional role of CAGE in cancer development, we generated stable HeLa transfectant clones in which the CAGE gene is overexpressed in the presence of tetracycline. As a result, CAGE overexpression increases the protein levels of the G1 cyclins, cyclin D1 and cyclin E. Cell synchronization experiments showed that CAGE overexpression promotes cell cycle progression. The inducing activity of CAGE for the expression of G1 cyclins was observed at the transcriptional level and even when cell growth was arrested by confluent cell growth, indicating that CAGE plays a role in up-regulating the transcription of the cyclin D1 and cylin E genes. Meanwhile, CAGE overexpression increased phosphorylation level of Rb at Ser-795, a site subjected to phosphorylation by both cdk2 and cdk4. Also, CAGE overexpression increased expression of E2F-1 and its release from Rb complex. Promoter analysis and gel mobility shift assay illustrated the functional involvement of AP-1/ E2F in CAGE-induced cyclcin D1/E expression. siRNA Knock-down of Jun D/E2F-1 abolished CAGE-mediated cyclin D1/E/A expression. These results indicate that CAGE promotes cell cycle progression by up-regulating expression of cyclcin D1 and cyclin E in E2F/AP-1-dependent manner.

목차 (Table of Contents)

• I. 서론 = 1
• II. 실험재료 및 방법 = 5
• 1. 세포배양 = 5
• 2. CAGE가 과발현 되었을 때 cyclin의 발현수준 분석 = 5
• 3. Histone H1 kinase assay = 6
• 4. Promoter assay = 7
• 5. Electrophoretic mobility shift assay(EMSA) = 7
• 6. Analysis of Activation of AP-1 family = 7
• 7. Transfection of siRNA = 8
• 8. BrdU Incorporation assay = 8
• III. 결과 = 10
• 1. HeLa/CAGE transfectant에서 cyclin의 발현수준 비교 = 10
• 2. BrdU incorporation assay를 통한 세포주기 조사 = 10
• 3. Histone H1 Kinase assay를 통한 cdk 인산화 활성 조사 = 10
• 4. CAGE가 과발현 되었을 때 Rb/E2F 발현 수준 비교 = 11
• 5. Cyclin 발현 수준을 조절하는 전사인자의 조사 = 12
• 6. CAGE가 과발현 될 때 AP-1 family의 발현수준 조사 = 12
• 7. siRNA를 이용한 Cyclin 발현 수준 조사 = 13
• IV. 논의 = 14
• V. 결론 = 18
• VI. 참고문헌 = 19
• Abstract = 32