Effects of SHIP gene mutation on cell cycle related proteins and phosphorylated Akt in K562 cells.
- Author:
Lin YANG
1
;
Jian-min LUO
;
Xiao-jun LIU
;
Shu-peng WEN
;
Jing-ci YANG
;
Jing-yu ZHANG
Author Information
- Publication Type:Journal Article
- MeSH: Cell Cycle Proteins; metabolism; Humans; Inositol Polyphosphate 5-Phosphatases; K562 Cells; Mutation; Phosphoric Monoester Hydrolases; genetics; Phosphorylation; Proto-Oncogene Proteins c-akt; metabolism; Transfection
- From: Chinese Journal of Hematology 2009;30(8):548-552
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo investigate the effect of SHIP gene mutation on the cell cycle and its related gene expression in K562 cells.
METHODSThe 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.
RESULTSWild 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).
CONCLUSION(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.