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- 저자 : 최운만 ( Choi Woon Min ) (검색결과 1 건)
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OVCAR-3와 SKOV-3 난소암 세포주에서 Flow Cytometry를 이용한 Apoptosis와 p53 단백의 측정
윤주희 ( Yoon Joo Hee ),최운만 ( Choi Woon Min ),조윤성 ( Jo Yun Seong ),나종구 ( Rha Jong Gu ),한구택 ( Han Gu Taeg ) 대한산부인과학회 2003 Obstetrics & Gynecology Science Vol.46 No.7
Objective : Taxol (paclitaxel)-induced apoptosis was studied to understand their biological mechanism correlated with the expression of p53 in the SKOV-3 and OVCAR-3 ovarian cancer cell lines. Materials and Methods : The SKOV-3 and OVCAR-3 cell lines were cultured in RPMI 1640 medium without taxol (control group) and with taxol for 24 h and 48 h (experimental group). After harvest, the cells were stained with annexin V-FITC (fluorescein isothiocyanate) and anti-cytokeratin antibodies (clone CAM5.2 and clone MNF116). They were washed and stained with p53 antibody. After then the secondary antibodies, i.e., FITC- or phycoerythrin (PE)-conjugated goal anti-mouse (GAM) immunoglobulin G (GAM IgG-FITC or GAM IgG-PE) were added in the cells and they were incubated in the dark. DNA of these cells were stained sequentially with propidium iodide (PI). Standard FASCcan equipped with a 499 ㎚ single laser was used for the analysis of these cells. Results : Both of SKOV-3 and OVCAR-3 cell lines were arrested in the G_2M phase after treatment of taxol, suggesting that these cells would eventually enter into the stage of cell death. Fractions of negative cytokeratin and positive annexin V and amount of sub-G_0G_1 fraction indicative of apototic fractions were lower in the SKOV-3 cell line compared with that in OVCAR-3 cell line, probably as a result of lower sensitivity of SKOV-3 cell line to the taxol. p53 expression were not detected in SKOV-3 cell line. On the basis of observed findings in SKOV-3 cell line and findings of high expressions of p53 regardless of taxol treatment, no increase in their expressions according to culturing time, and gradual increases in sub-G_0G_1 fractions and in fractions of negative cytokeratin and positive annexin V indicative of apoptosis in OVCAR-3 cell line, we concluded that the expression of p53 would not be associated with cell cycle changes and the arrest in the G_2M pahse but associated with the appearance of apotosis. Conclusion : Our results suggest that flow cytometric detection of the apoptotic fractions would be an effective, fast, and accurate method for the chemosensitivity test in tumor cells before the administration of anti-cancer drugs in gynecologic cancer patients.
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