[Analysis of cell cycle checkpoints by cyclins/DNA flow cytometry].
OBJECTIVE
Eukaryotic cell cycle events progress strictly in order which is controlled by the mechanism of checkpoint. At present, most analyses of checkpoint use the flow cytometry (FCM) based on DNA histogram to detect cell cycle distribution. This study was designed to set up and evaluate a new method, Cyclins/DNA multiparameter FCM based on the model of late G1 phase (G1L) checkpoint, for analyzing cell cycle checkpoint.


METHODS
After irradiation by ultraviolet (UV), human acute lymphatic leukemia MOLT-4 cells were gathered and fixed at different time points, and divided into 2 groups. In one group, the total G0/G1 phase cells were calculated by Modifit software using DNA histogram method; in the other group, fluorescence intensity and threshold of Cyclin E in G1L cells and G0/G1 phase cells were quantitatively analyzed by Cyclins/DNA multiparameter method.


RESULTS
When analyzed by DNA histogram method, the percentage of G0/G1 phase cells was unchanged after irradiated for 0-4 h, but increased to 12.6% after irradiated for 6 h. When analyzed by Cyclins/DNA multiparameter method, the Cyclin E fluorescence intensity of G1L cells was increased from 295.1 (control) to 341.2 (15.6%) after irradiated for 1 h, and increased to 577.6 (95.7%) with the threshold increased from 2.0 (control) to 5.4 after irradiated for 6 h; G1L cells was slightly decreased after irradiated for 6 h when the apoptosis rate was 5.61%, and early G1 phase (G1E) cells was increased slowly.


CONCLUSIONS
Cyclin E/DNA multiparameter FCM could quantitatively detect fluorescence intensity and threshold of Cyclin E, and is more sensitive and precise than DNA histogram FCM in detecting G1L checkpoint.
Xie
Da-Xing
DX
Cancer Research Institute, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, P.R. China.
Wu
Jian-Hong
JH
Feng
Yong-Dong
YD
Li
Xiao-Lan
XL
Tao
De-Ding
DD
Gong
Jian-Ping
JP
D002453:Cell Cycle; D045744:Cell Line, Tumor / Q000528:radiation effects; D049109:Cell Proliferation; D019927:Cyclin E / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry; D015458:Leukemia, T-Cell / Q000378:metabolism / Q000473:pathology*; D014466:Ultraviolet Rays
D019927:Cyclin E; D004247:DNA