- 사용 시약과 기기 - 세포주 - 985

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1 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

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,

2 - 사용 시약과 기기 - 세포주 - 985

3 - 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

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.

4 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

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.

5 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 Korean J Otolaryngol 2002;45:984-9

observed. However, for 5-FU IC 50 concentrations, cells accumulated in G0/G1 phase.

6 REFERENCES 1) Varvares MA, Cheney ML. Reconstruction of the hypopharynx and cervical esophagus. In cummings CW (eds): Otolaryngology head & neck surgery. 3rd ed., St. Louis, Mosby Year Book;1998. p ) Beauvillain C, Mahe M, Bourdin S, Peuvrel P, Bergerot P, Riviere A. Final results of a randomized trial comparing chemotherapy plus radiotherapy with chemotherapy plus surgery plus radiotherapy in locally advanced resectable hypopharyngeal carcinoma. Laryngoscope 1997;107: ) Kim KH, Sung MW, Koo JW, Lee DW, Moon BK, Lee CH. Neoadjuvant chemotherapy and radiotherapy for the treatment of advanced hypopharyngeal carcinoma. Korean J Otolaryngol 1997; 40: ) Wang X, Wong SCH, Pan J, Tsao SW, Fung KHY, Kwong DLW, Sham JST, Nicholls JM. Evidence of cisplatin-induced senescentlike growth arrest in nasopharyngeal carcinoma cells. Cancer Research 1998;58: ) Jacobs C, Lyman G, Velez-Garcia E, Sridhar KS, Knight W, Hochster H, Goodnough LT, et al. A phase III randomized study comparing cisplatin and fluorouracil as single agents and in combination for advanced squamous cell carcinoma of the head and neck. J Clin Oncol 1992;10: ) Jacobs C, Bertino JR, Goffinet DR, Fee WE, Goode RL. 24-hour infusion of cis-platinum in head and neck cancers. Cancer 1978; 42: ) Al-Sarraf M. Head and neck cancer: chemotherapy concepts. Semin Oncol 1988;15: ) Ensley JF, Kish JA, Weaver AA, Jacobs JR, Hassan M, Cummings G, et al. The correlation of specific variables of tumor differentiation with response rate and survival in patients with advanced head and neck cancer treated with induction chemotherapy. Cancer 1989; 63: ) O Conner PM, Jackman J, Bae I, Myers TG, Fan S, Mutoh M, et al. Characterization of the p53 tumor suppression pathway in cell lines of the national cancer institude anticancer drugs screen and correlations with the growth-inhibitory potency of 123 anticancer agents. Cancer Research 1997;57: ) Roh HJ, Goh EK, Wang SG, Chon KM, Kim YS, Han JY. Establishment and characterization of a novel cell line (PNUH-12) derived from a human squamous cell carcinoma of the hypopharynx. Korean J Otolaryngol 1999;42: ) Tokino T, Nakamura Y. The role of p53-target genes in human cancer. Critical Reviews in Oncology Hematology 2000;33: ) Mueller H, Eppenberger U. The dual role of mutant p53 protein in chemosensitivity of human cancers. Anticancer Research 1996;16: ) Koechli OR, Schaer GN, Seifert B, Hornung R, Haller U, Eppenberger U, et al. Mutant p53 protein associated with chemosensitivity in breast cancer specimens. The Lancet 1994;344: ) Zamble DB, Jacks T, Lippard SJ. P53-dependent and -independent responses to cisplatin in mouse testicular teratocarcinoma cells. Proc Natl Acad Sci USA 1998;26:95: ) Mirjolet JF, Barberi-Heyob M, Didelot C, Peyrat JP, Abecassis J, Millon R, et al. Bcl-2/Bax protein ratio predicts 5-fluorouracil sensitivity independently of p53 status. Br J Cancer 2000;83: ) Patel V, Ensley JF, Gutkind JS, Yeudall WA. Induction of apoptosis in head-and-neck squamous carcinoma cells by gamma-irradiation and bleomycin is p53-independent. Int J Cancer 2000;88: ) Wagner AJ, Kokontis JM, Hay N. Myc-mediated apoptosis requires wild-type p53 in a manner independent of cell cycle arrest and the ability of p53 to induce p21waf1/cip1. Genes Dev 1994 Dec 1;8: ) Siddik ZH, Mims B, Lozano G, Thai G. Independent pathway of p53 induction by cisplatin and X-rays in a cisplatin-resistant ovarian tumor cell line. Cancer Research 1998;58: ) Yoneda K, Yamamoto T, Osaki T. p53- and p21-independent apoptosis of squamous cell carcinoma cells induced by 5-fluorouracil and radiation. Oral Oncol 1998;34: ) Bissonnette N, Hunting DJ. p21-induced cycle arrest in G1 protects cells from apoptosis induced by UV-irradiation or RNA polymerase II blockage. Oncogene 1998;16:

Final results of a randomized trial comparing chemotherapy plus radiotherapy with chemotherapy plus surgery plus radiotherapy in locally advanced resectable hypopharyngeal carcinoma.

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( )Jkstro011.hwp 비인강암의방사선치료결과및생존율에관한예후인자분석 2005 2 1 2005 3 28. :, Tel: 053)250-7665, Fax: 053)250-7984 E-mail: jhkim@dsmc.or.kr 정영연외 2 인 : 비인강암의예후인자분석 정영연외 2 인 : 비인강암의예후인자분석 Carcinoma of the nasopharynx treated by radiotherapy