[Detection of cell cycle related gene expression in cultured immortalized human lens epithelial cell].
OBJECTIVE
To investigate the gene expression of cyclin E, cyclin-dependent-kinase 2 (CDK(2)) and CDK inhibitor (CKI) p21 during cell cycle of the human lens epithelial cell (HLEC) for elucidating the molecular mechanism of proliferation of HLEC and the formation of posterior capsular opacification (PCO).


METHODS
HLE-B3 cell line was cultured in vitro under MEM medium with 15% fetal bovine serum. The cell cycle was analyzed by flow cytometry (FCM) system. The cell cycle was synchronized by double thymidine block method. The mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). The total RNA was separated from cells at different phases. Protein expression was detected with immunohistochemistry or western blotting method. The primary antibodies were rabbit anti-human cyclin E polyclonal antibody, mouse anti-human p21 and CDK(2) monoclonal antibody, respectively.


RESULTS
FCM showed that G(1) phase was about 33.5%, S phase cells about 46.1%, and the G(2) phase about 20.4%, total cells in G(2) and S phase were over 60%. It was suggested that HLE-  B3 cells have relatively more capacity of mitosis than normal LEC. The mRNA expression of CDK(2) was found at all phases of cell cycle during synchronization, but cyclin E mRNA only expressed during synchronized cells, and not expressed in non-synchronized cells. Only Sphase cells could express very low p21 mRNA. There was no any positive signal of cyclin E by immunohistochemistry or western blot. CDK(2) protein detected by Western blot expressed at all phases. The p21 protein was expressed in a few cells by immunohistochemistry, but p21 could not detected by western blotting. These results showed cyclin E and CDK(2) were higher expressed than p21 in transcriptional level and translational level.


CONCLUSIONS
There is mRNA expression of cyclin E, CDK(2) and p21 during the cell cycle in HLE-B3 cell line, but it means the imbalance of the association of CDK and CKI. These results suggest that the imbalance expression of cyclin, CDK and p21 be the molecular mechanism of PCO.
Wu
Mingxing
M
Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China. wumx63@21cn.com
Li
Shaozhen
S
Zeng
Junwen
J
Gao
Jinsong
J
Liu
Yizhi
Y
D015153:Blotting, Western; D042846:CDC2-CDC28 Kinases; D002386:Cataract / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics*; D002460:Cell Line; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D005434:Flow Cytometry; D016193:G1 Phase / Q000235:genetics; D016195:G2 Phase / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase / Q000235:genetics
D019927:Cyclin E; D012333:RNA, Messenger; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases