Detection of Apoptosis by the Stainings of Annexin V, Propidium Iodide and Cytokeratin in OVCAR-3 Ovarian Cancer Cell Line

Song, Min Kyung; Kwon, Moon Ki; Lee, Jung Woong; Choi, Ye Hoon; Kim, Tae Woo; Ryu, Ki Sung; Rha, Jong Gu; Han, Ku Taek

Abstract

OBJECTIVE
This study was designed to estimate the chemosensitivity by a quantitative evaluation of the apoptotic cell fractions using flow cytometry.
METHODS
The OVCAR-3 cells were exposed to 20 nM or 30 nM taxol for 0 (control), 24 and 48 hours, then removed the taxol contained media, and cultured further with fresh media without taxol. (1) Fluorescein isothiocyanate-conjugated Annexin V (Annexin V-FITC) and propidium iodide (PI) were added to one test tube to detect the apoptotic cell fractions and at the same time, PI was added to the other tube to stain the DNA. (2) Annexin V-FITC and cytokeratin (clone CAM5.2 and MNF116) were added to the test tube. They were fixed and permeabilized with 1% paraformaldehyde solution and 100% methanol. They were then incubated with phycoerythrin (PE)-conjugated goat anti-mouse immunoglobulin G (GAM IgG1-PE or GAM IgG2a-PE) and sequentially stained with PI for DNA. All the stained cells were analyzed by a FACScan flow cytometer.
RESULTS
(1) After treatment of 20 nM or 30 nM of taxol, G2M arrest was observed in both of treatment groups, which increased with time. (2) The G0G1 sub-fraction indicative of apoptosis increased with increase of culturing time from 24 hrs to 48 hrs. (3) The early apoptotic cell fraction with positive annexin V-FITC and negative PI increased with increase of culturing time. (4) In cells stained sequentailly with annexin V-FITC, cytokeratin (CAM5.2 and MNF116), and PI after 30 nM taxol treatment, the early apoptotic cell fractions increased with increase of culturing time. However, their extent was somewhat lower than those observed by positive annexin V-FITC and negative PI in cells treated with 20 nM of taxol.
CONCLUSION
The results of sequential stainings with annexin V-FITC, cytokeratin, and PI were consistent with the those of annexin V-FITC and PI with parallel DNA staining. Our results suggested that the level of apoptosis detected by flow cytometry could be a marker of chemosensitivity which could select the sensitive anti-cancer agents before administration to gynecologic cancer patients.