[Effects of SHIP gene mutation on cell cycle related proteins and phosphorylated Akt in K562 cells].
OBJECTIVE
To investigate the effect of SHIP gene mutation on the cell cycle and its related gene expression in K562 cells.


METHODS
The recombined green fluorescent protein (GFP) containing FIV-SHIP gene was transfected into K562 cells. The transfection efficiency and cell cycle of K562/SHIP were assessed by flow cytometry (FCM). The proliferation of K562 cells was detected by MTT assay, the mRNA levels of SHIP by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR), and the protein levels of SHIP, Cyclin D1, p21(WAF1/CIPI) and p27(KIP1) by Western blot.


RESULTS
Wild type SHIP inhibited K562 cell proliferation and caused a G(0)/G(1) arrest \[(34.2 +/- 7.8)% vs (0.7 +/- 8.3)% (P < 0.01)\]; while the point mutation of SHIP gene did not show such effect. Western blot results showed that the Akt phosphorylation and cyclin D1 expression was significantly decreased (P < 0.01), and the expression of p27(KIP1) and p21(WAF1/CIPI) increased. Site-directed mutation of SHIP gene SH2 domain (TTC-->CTC, Phe-->Leu) did not influence the Akt phosphorylation and cyclins (P > 0.05).


CONCLUSIONS
(1) wtSHIP gene can down-regulate Akt phosphorylation and result in inhibition of cyclin D1 expression, up-regulating p27(KIP1) and p21(WAF1/CIPI) expression, finally leading to the reduction of K562 cell proliferation, and inducing G(0)/G(1) phase arrest. (2) SHIP gene suppresses the proliferation of K562, being dependent on its intact structure and function.
Yang
Lin
L
Department of Hematology, the 2nd Hospital of Hebei Medical University, Shijiazhuang 050000, China.
Luo
Jian-min
JM
Liu
Xiao-jun
XJ
Wen
Shu-peng
SP
Yang
Jing-ci
JC
Zhang
Jing-yu
JY
D018797:Cell Cycle Proteins / Q000378:metabolism*; D006801:Humans; D000072181:Inositol Polyphosphate 5-Phosphatases; D020014:K562 Cells; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D014162:Transfection
D018797:Cell Cycle Proteins; D051057:Proto-Oncogene Proteins c-akt; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases