[Experimental study of up-regulating PTEN gene on increasing the chemosensitivity in K562/ADM cells].
OBJECTIVE
To investigate the mechanism of decreasing the K562/ADM cells chemosensitivity by up-regulating expression of PTEN gene.


METHODS
K562/ADM cells were transient transfected with pGFP-PTEN or vector. The level of PTEN in K562/ADM cells was assayed by Western blot and RT-PCR. Cell viability on K562/ADM was determined by MTT assay. Cell apoptosis by flow cytometry. Activity of caspase-3 by Caspase Colorimetric Assay Kit. The proteins expression of LC3-I/II, Beclin1, p-Akt, p-p70S6K by Western blot. The autophagic vacuoles by MDC stain and Electron microscopy.


RESULTS
(1) The mRNA and protein levels of PTEN in K562/ADM cells transfected with pGFP-PTEN were significantly increased compared with the control (untreated and transfected with empty vector). (2) Enhanced expression of PTEN by gene transfection resulted in a reversal of resistance to ADM. Compared with empty vector group, cell viability decreased from (94.07 ± 2.6)% to (53.83 ± 4.2)%, the cell apoptotic rate increased from (11.89 ± 1.7)% to (43.69 ± 2.3)%, meanwhile, pretreated with caspase-3 inhibitor (Z-DEVE-FMK) didn't completely inhibit the cytotoxicity of ADM to K562/ADM cells. (3) After treated with ADM for 12 and 24 h, the activities of caspase-3 in PTEN-transfected K562/ADM cells increased compared with those in pGFP-transfected K562/ADM cells \[(2.27 ± 0.13) vs (1.19 ± 0.14)\] at 12h, \[(3.15 ± 0.08) vs (1.48 ± 0.05)\] at 24 h (P < 0.05). (4) The protein levels of LC3-II and Beclin1 in K562/ADM cells transfected with pGFP-PTEN were increased by 83% and 18% respectively, and the protein levels of p-Akt and p-p70S6K were declined by 96% and 87% respectively, compared with those in K562/ADM cells transfected with pGFP plasmid. (5) The upregulation of PTEN in K562/ADM cells improved the number of autophagic vacuoles compared with the empty vector group.


CONCLUSIONS
The upregulation of PTEN expression increases the chemosensitivity of K562/ADM to ADM, which may related with the inhibition of PI3K/AKT/mTOR pathway induced by PTEN gene transfection.
Department of Pediatrics, Xiangya Hospital, Central South University, Changsha, China.
Hong
H
Zhang
Liang-chun
LC
Yang
Li-zhi
LZ
Cao
Ming-hua
MH
Yang
Min
M
Xie
Shan
S
Zhu
Pei
P
Zeng
Yan
Y
Yu
D017209:Apoptosis; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D015973:Gene Expression Regulation, Leukemic; D005822:Genetic Vectors; D006801:Humans; D020014:K562 Cells; D007938:Leukemia / Q000188:drug therapy*; D051059:PTEN Phosphohydrolase / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D015854:Up-Regulation
D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human