KISEP Head and Neck Korean J Otolaryngol 2002;45:984-9 하인두 편평상피암 세포주(PNUH-12)에서 Cisplatin 및 5-Fluorouracil 처리 후 p53의 Molecular Event 변화에 대한 연구 2 전경명 1 이병주 1 이일우 1 문영일 1 노환중 1 왕수건 1 고의경 1 김소린 1 이은엽 The Change of Molecular Event of p53 by Cisplatin and 5-Fluorouracil in Hypopharyngeal Cell Line PNUH-12 Kyong-Myong Chon, MD 1, Byung-Joo Lee, MD 1, II-Woo Lee, MD 1, Young-Il Moon, MD 1, Hwan-Jung Roh, MD 1, Soo-Geun Wang, MD 1, Eui-Kyung Goh, MD 1, So-Rin Kim, BS 1 and Eun-Yup Lee, MD 2 1 Department of Otolaryngology and 2 Clinical Pathology, College of Medicine, Busan National University, Busan, Korea ABSTRACT Background and ObjectivesIn head and neck cancer including hypopharyngeal carcinoma, cisplatin and 5-fluorouracil usually have been used as neoadjuvant chemotherapeutic agents. We investigated the difference in the influences of cisplatin and 5- fluorouracil 5-FU on the p53 protein expression and cell responses cell cycle arrest and apoptosis in the hypopharyngeal cell line PHUH-12. MethodPNUH-12 with a mutant type p53 one point mutation at the 78th base, C to G, in exon 7 was treated with cisplatin and 5-FU. Changes in the cell line were assessed by MTT assay, Western blotting p53 and p21 protein, DNA fragmentation, PI stain, and DNA flow cytometry. ResultsThe p53 protein expression was increased after the treatment with cisplatin and 5-FU. The expression of p21 protein was increased after the treatment with 5-FU, not cisplatin. With cisplatin, we observed apoptosis by DNA fragmentation and PI stain and the increased S phase on DNA flow cytometry. But, with 5-FU, we couldn t observe apoptosis by DNA fragmentation, PI, and flow cytometry and only the increased G1 phase on DNA flow cytometry. ConclusionIn hypopharyngeal cell line PNUH-12, cisplatin induced p53 dependent apoptosis and 5-FU induced p53 and p21 dependent G0/G1 cell cycle arrest, but not apoptosis. Korean J Otolaryngol 2002;45:984-9 KEY WORDSCisplatin Fluorouracil Hypopharyngeal neoplasms. 984 - -
- 사용 시약과 기기 - 세포주 - 985
- 986 A B Fig. 1. Cytotoxicity of cisplatin A and 5-FU B in PHUH-12 cells with cell growth determined by MTT assay. As the concentration and exposure time of the cisplatin and 5-FU increased, the survival decreased. Fi 2 W t bl t l i i f 53 t i d Fig. 2. Western blot analysis expression of p53 protein and p21 protein in PNUH-12 cell after the exposure of cisplatin and 5-FU. p53 protein increased in the exposure of both cisplatin 3 M, 12 hr and 5-FU 30 M, 12 hr. But in the treatment of cisplatin, the increase of p21 protein was not observed. But in the treatment of 5-FU, the increase of p21 protein was observed. Korean J Otolaryngol 2002;45:984-9
Fig. 3. DNA electrophoresis. Control groups lane A and E don t show DNA fragmentation. Cells treated with cisplatin lane C and D shows typical DNA laddering patterns. Cells treated with 5-FU lane F, G, and H don t show DNA laddering patterns. A and Econtrol groupsb, C, and D4 M, 8 M, and 16 M for 48 hours, respectivelyf, G, and H 40 M, 80 M, and 160 M for 48 hours, respectively. A B Fig. 4. Propidium iodide stain of control A and 8 M 48 hours cisplatin induced apoptotic PNUH-12 cell B. Control shows homogeneous staining of their nuclei. In contrast, apoptotic cells show irregular staining of nuclei due to results of chromatin condensation and nuclear fragmentation. - - 987
A B C Fig. 5. DNA flow cytometry. For cisplatin IC 50 concentrations, a gradual increased of cells in S phase was observed. However, for 5-FU IC 50 concentrations, cells accumulated in G0/G1 phase. Acontrol. B24 hours after incubation with 3 M cisplatin. C24 hours after incubation with 30 M 5-FU. - 988 Korean J Otolaryngol 2002;45:984-9
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