[Down-regulation of Chk1/Chk2 gene expression increases apoptosis in irradiated HeLa cells and its mechanism].
OBJECTIVE
To explore the increasing effect of blocking Chk1 and /or Chk2 gene by Chk1 or Chk2-specific antisense oligodeoxynucleotides (AsODN) on apoptosis in HeLa cell line after irradiation and its mechanism of action.


METHODS
Asynchronized HeLa cells were exposed to (60)Co-irradiation at different dosage to activate G(2)/M checkpoint arrest. The cell cycle profiles were observed in HeLa cells after irradiation at a range of various doses and different time points by flow cytometry. In the experimental groups, Chk1/2 sODN and AsODN alone or in combination were transfected into HeLa cells, and the cells were exposed to (60)Co-irradiation at 24 h after transfection. The changes of Chk1/2 protein expression were assayed by Western blot and confocal laser scanning microscopy (Confocal), and the cell cycles, apoptosis rates and cell cycle specific apoptosis were detected by annexin V-PI labeling and flow cytometry.


RESULTS
Apoptotic response was significantly increased in the Hela cells after G(2)/M arrest and was inversed to activation of G(2)/M checkpoint. Either Chk1 or Chk2-specific AsODN consistently enhanced DNA damage-induced apoptosis by 90% approximately 120%, compared to corresponding sODN control (P < 0.05). Unexpectedly, combined use of Chk1- and Chk2-specific AsODN did not produce synergistic effect as compared to treatment with Chk1- or Chk2-specific AsODN alone (P > 0.05). While irradiated HeLa cells underwent apoptosis preferentially in G(1)-phase, apoptosis occurred in either of G(1)-, S- or G(2)/M -phase in the presence of Chk1 and/or Chk2 AsODN.


CONCLUSIONS
The radioresistance is mainly induced by activating the cell cycle checkpoint signal transduction pathway after irradiation, and abrogating of the key effector Chk1 and Chk2 may increase the apoptotic sensitivity to irradiation due to changes of the pattern of cell cycle specific apoptosis.
Gao
Qing-lei
QL
Department of Gynecology & Obstetrics, Tongji College of Huazhong Science & Technology University, Wuhan 430030, China.
Ye
Fei
F
Xing
Hui
H
Xie
Da-xing
DX
Lu
Yun-ping
YP
Zhou
Jian-feng
JF
Ma
Ding
D
D017209:Apoptosis / Q000528:radiation effects; D002453:Cell Cycle / Q000528:radiation effects; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D003037:Cobalt Radioisotopes; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D003037:Cobalt Radioisotopes; D020319:Oligodeoxyribonucleotides, Antisense; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases