Abstract 5050

Introduction

SHIP is an SH2 domain containing inositol-5-phosphatasse that appears to be a negative regulator of hematopoiesis. Our previous researching works have first reported that SHIP gene was genetically altered in leukemia patients and proved that mutation of SHIP gene was closely correlated with the increased phosphorylation of Akt and poor prognosis of AML patients; SHIP gene is a key regulative gene in the PI3K/Akt pathway, and transfection with wild type SHIP gene into K562 cell line can inhibit the proliferation of K562. Up to now, however, there are no any reports about what changes of the PI3K/Akt signal transduction regulation will happened if SHIP has site mutation in leukemia cells. The objectives of our study is to investigate the effects of site-directed mutation of SHIP on the expression level of the protein related to the PI3K/Akt signal pathway in leukemia cells, and to study the effects of site-directed mutation of SHIP on the key PIP3 activity related to the PI3K/Akt signal pathway in leukemia cells.

Methods

Based on the results of our previous studies, the absence of endogenous SHIP in K562 cells provided a useful systerm to study the role of SHIP in growth and apoptosis. The recombined lentivirus plasmids wtSHIP or muSHIPP28L were transfected stably into human leukemia cells K562. The cell proliferation, cell life cycle and cell apoptosis of K562 transfected with wid-type SHIP or muSHIPP28L were determined by MTT, fluorescent staining and flow cytometry. The expression level and difference of total Akt□Ap-Akt473 and p-Akt308 were reconfirmed by SDS-PAGE western blot. PI(3,4,5)P3 and PI(3,4)P2 was assayed by High pressure liquid chromatography.

Results

The decreased ability of proliferation and DNA synthesis, cell colony fomation ability and enhanced apoptosis rate were observed in K562 cells transfected with wild-type SHIP (Group A), but the same changes had not been observed in K562 cells transfected with muSHIPP28L (Group B) or empty vector (Group C). Wild-type SHIP can down-regulate phosphorylations of Akt308 and Akt473, but muSHIPP28L can't. High pressure liquid chromatography results showed that the PI3,4,5-P3 level was obviously decreased in Group A,no changes above indicate in Group B and Group C. The PI3,4P2 level in Group A was significantly higher than Group B and Group C.

Conclusions

The results confirmed SHIP as a negative regulator for cell proliferation in leukemia cells, and implied that it may function through its normal structure.

Disclosures

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.

Sign in via your Institution