[Suppression of HCT116 growth of cells by Gadd45].
OBJECTIVE
To study the mechanism of suppression of growth of HCT116 human colon carcinoma cells by Gadd45 gene.


METHODS
HCT116 human colon carcinoma cells were transfected with pTRE-Gadd45 vector so as to establish Gadd45-inducible cell line that was cultured in medium with tetracycline. Then tetracycline was withdrawn. The number of cell clones was counted. Flow cytometry was used to detect the percentages of cells at the G1, S, and G2-M phases. TUNEL technique was used to detect the apoptosis of cells. Western blotting was used to analyze the expression of Gadd45 protein, and apoptosis-related proteins: poly-ADP-ribose polymerase (PARP), and caspase 3 protein.


RESULTS
Gadd45 protein was not expressed in the HCT116 cells cultured in the medium with tetracycline, however, it was expressed with a gradually increased level in the cells cultured in the medium from which tetracycline was withdrawn, The clone formation rate of HCT116 cells was 100% in the medium with tetracycline, however, was only 14.2% in the medium with tetracycline withdrawal, with a suppression rate of more than 85%. The percentage of cells in G(2)-M phase was significantly increased in the cells cultured in the medium with tetracycline withdrawal. 24-36 hours after the withdrawal of tetracycline, PARP and caspase 3 protein were activated remarkably.


CONCLUSIONS
High expression of Gadd45 inhibits the growth of HCT116 cells, through inducing G2-M arrest and activating apoptotic pathway.
Tong
Tong
T
State Key Laboratory of Molecular Oncology, Cancer Institute, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100021, China.
Song
Yong-mei
YM
Fu
Ming
M
Dong
Li-jia
LJ
Jin
Shun-qian
SQ
Wu
Min
M
Zhan
Qi-min
QM
D015153:Blotting, Western; D002455:Cell Division; D003110:Colonic Neoplasms / Q000517:prevention & control*; D005434:Flow Cytometry; D016195:G2 Phase; D045325:HCT116 Cells; D006801:Humans; D020287:In Situ Nick-End Labeling; D047908:Intracellular Signaling Peptides and Proteins; D008938:Mitosis; D011506:Proteins / Q000502:physiology*; D013752:Tetracycline / Q000494:pharmacology; D000097642:GADD45 Proteins
D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D013752:Tetracycline; D000097642:GADD45 Proteins