Loss of Cyclin B1 followed by downregulation of Cyclin A/Cdk2, apoptosis and antiproliferation in Hela cell line.
Recent studies have shown that Cyclin B1 is overexpressed in various tumor types but present at low levels in normal tissues. To explore the possibility of employing Cyclin B1 as an anticancer target, we knocked down Cyclin B1 in an HeLa cell line using RNA interference (RNAi). Subsequently, we monitored cell cycle-related molecules by Western blot together with immunofluorescence and determined cell cycle distribution by flow cytometry. XTT and soft agar colony growth experiments were performed to detect cell viability and proliferation. Furthermore, we analyzed cell apoptosis by measuring Bcl-2 and Bax protein level and DNA-ladder assay. After performing Cyclin B1 RNAi, Cyclin B1, Cyclin A and Cdk2 protein levels were found to be markedly downregulated, whereas Cdc2 was almost unaffected; S-phase fraction increased significantly; HeLa cell viability and cell colony forming ability were markedly diminished after the RNAi; Bcl-2 was noticeably attenuated but Bax was hardly changed; and HeLa cells displayed typical DNA ladder. The loss of Cyclin B1 resulted in the downregulation of Cyclin A and Cdk2, S-phase delay and eventually led to cell apoptosis and the decrease of cell viability and proliferation. Our studies suggest that Cyclin B1 may be a promising anticancer target.
Xie
Xian He
XH
Brain Korea 21 Project for Medical Science, Yonsei Univesity, Seoul, Korea.
An
Hee Jung
HJ
Kang
Suki
S
Hong
Sunghui
S
Choi
Yoon Pyo
YP
Kim
Young Tae
YT
Choi
Young Deuk
YD
Cho
Nam Hoon
NH
D017209:Apoptosis; D015153:Blotting, Western; D042846:CDC2-CDC28 Kinases / Q000096:biosynthesis*; D049109:Cell Proliferation; D002470:Cell Survival; D019925:Cyclin A / Q000096:biosynthesis*; D019926:Cyclin B / Q000235:genetics* / Q000502:physiology*; D056744:Cyclin B1; D051357:Cyclin-Dependent Kinase 2; D015536:Down-Regulation; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D034622:RNA Interference
C534699:CCNB1 protein, human; D019925:Cyclin A; D019926:Cyclin B; D056744:Cyclin B1; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2