[Silencing of cell cycle checkpoint kinase gene enhances cisplatin-induced apoptosis of lung cancer cells].
OBJECTIVE
To investigate the changes in cell cycle induced by cisplatin (DDP) and the effect of antisense oligonucleotide (AsODN) targeting Chk1/2 on DDP-induced apoptosis in lung cancer cell line A549 cells.


METHODS
The characteristics of cell cycle and apoptosis induced by DDP were detected by flow cytometry using SubG1 method. Chk1/2 mRNA and protein expression were assayed by RT-PCR and Western blot under best condition of transfection of AsODN targeting Chk1/2 by lipofection. Apoptosis of A549 cells induced by DDP was determined by flow cytometry using AnnexinV-FITC staining after transfection of Chk1/2 AsODN.


RESULTS
Asynchronized A549 cells were treated with 10 micromol/L DDP, and significant S-phase arrest was observed at 12 h later. Transfection with antisense oligonucleotide targeting Chk1/2 inhibited the Chk1/2 expression at both mRNA and protein levels. Either Chk1 or Chk2-specific AsODN consistently enhanced DNA damage-induced apoptosis by 100% - 200%, compared with that in the sODN control (P < 0.05), but combined use of Chk1- and Chk2-specific AsODN did not show synergistic effects as compared with that induced by treatment with Chk1- or Chk2-specific AsODN alone (P > 0.05).


CONCLUSIONS
Chk1 and Chk2 may be regarded as effective targets of chemotherapy for lung cancer. Silencing the key effector Chk1 and Chk2 genes may significantly increase the chemosensitivity of lung cancer cells.
Department of Surgery, Tongji Hospital of Huazhong Science & Technology University, Wuhan 430030, China.
Fei
F
Ye
Da-xing
DX
Xie
Yun-ping
YP
Lu
Qing-lei
QL
Gao
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D002945:Cisplatin / Q000494:pharmacology*; D020868:Gene Silencing; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D016376:Oligonucleotides, Antisense / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection
D000970:Antineoplastic Agents; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D002945:Cisplatin