Anti-leukemia Effects Of Wild SHIP Gene And Its Mechanism

Background: Leukemia is the most common malignant clone's disease of hematopoietic stem cells. The incidence rate of leukemia has the tendency of step-up recent years. Leukemia is the consequences of a multistep process resulting from a series of genetic changes and incidence, and its process is related with activation of oncogene or inactivation of antioncogene. Altohugh many genes'function and machanisms in leukemia has been explicit, all novel gene can not explain all pathogenesis of all leukemia. Thereby, it has become one of the hotspots to investigate leukemia by identifying new correlative oncogene or tumor suppressor genes and studying its biologic function,and it is important for elucidate the pathogenesy of leukemia.SH2-containing inositol 5'-phosphatase (SHIP) is a recently identified lipid phosphatase after phosphatase and tensin homologue deleted on chromosome 10(PTEN) was observed, which hydrolyzes the 5 prime phosphate of the inositol ring from inositol 3,4,5-P3. It is mainly expressed in hematopoietic cells. Its phosphatase activity toward PIP3 has been linked to suppress several signaling pathways, including Akt, leading to the inhibition of hematopoietic cell proliferation and survival. Our previous researching works have proved that low expression and deletion of SHIP gene was closely correlated with the abnormal proliferation of patients with leukemia; lower Akt activation was detected in K562 cell lines, which was caused by the re-expresion SHIP. transfection of wild type SHIP into the K562 cells can regulate the expression of proliferative relative genes and reverse the proliferative phenotype of K562. The molecular mechanism that SHIP gene suppress leukemia may be involved in some important signal pathways. Up to now, However, there are no any reports about the exact mechanisms of SHIP gene on the regulation of PI3K/Akt signal transduction pathway, the effects of SHIP gene on suppression and/or reversion of abnormal proliferation in human leukemia cell line K562 in the world. There are also no any reports about what changes of the PI3K/Akt signal transduction regulation will happened if SHIP has site mutation in K562 cell lines. The aim of this study are as follows: (1)To detect the influence of cell proliferation and apoptosis of K562 cells transfected with wild-type SHIP gene, and to investigate the molecular mechanism of apoptosis of K562 cells transfected with wtSHIP gene; (2) To construct SHIP gene's mutant (P28L) found in AML patients. (3) To investigate the functional changes of SHIP gene's mutant by over-expression it into human leukemia cell line K562,and to make a preliminary discussion on the mechanism of SHIP gene abnormality in leukemia.Part one: Effects of wild-type SHIP on proliferation, apoptosis and cell cycle of K562Objective To investigate in vitro the effects of SH2-containing inositol 5'-phosphatase gene-SHIP on proliferation, apoptosis, cell cycle of human leukemia cell line K562 and to explore its possible machinesim preliminarily.Method Based on the results of our previous studies, with different human leumemia cell lines K562 as study samples, including K562, K562/wtSHIP, K562/FIV; the growth of K562 was observed by MTT assay; the transfection efficiency and cell cycle distribution were assessed by flow cytometry (FCM). Morphological characteristics of light microscope,cell colony fomation,TUNEL and Hoechest 33342 fluorescent staining method were also used to test proliferation and apoptosis of K562 cells. The expression level and difference of SHIP mRNA was detected by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR); the the expression level and difference of SHIP, Akt, p-Akt473, p-Akt308 and the member of apoptosis family Bcl-2, Bcl-xL, Bax, Bad, cell cycle related molecular CyclinD1, p21WAF1 and p27Kip1 were detected by Western blot; Caspase-3, Caspase-9 protein activity were detected by caspase activity kits.Results1 The transfection efficiency was about 74.6±5.8% in K562 cell line after transfected with lentivirus on 48 h detected by flow cytometry. The recombinant lentivirus pReceiver-LV-SHIP has been successfully transfected into K562 cell line.2 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, and the same changes had not been observed in K562 cells transfected with empty vector and untransfected K562(P<0.01).3 Cell cycle distribution showed the ratio of G0/G1 phase of K562 cells transfected with wild-type SHIP gene increased from (34.15±8.30)% to (60.69±8.30)%, the ratio of G2/M+S phase decreased form(65.85±9.36)% to (38.85±7.09)%, the cell cycle was arrested at G0/G1 phase.4 After transfection wtSHIP at the third days, western blot results showed the Akt expression levels had no change but p-Akt308 and p-Akt473 expression level (0.125±0.016 and 0.351±0.064 respectively) decreased compared with K562/FIV group ( Akt308 : 0.323±0.025 , Akt473 :0.788±0.102) and untransfected group(Akt308:0.324±0.023,Akt473:0.801±0.095)( P<0.01).5 wtSHIP gene can inhibit the proliferation of K5626 After K562 cells transfected wtSHIP, the cell cycle related molecular expression levels showed that the protein expression levels of p27,p21 (0.302±0.059,0.335±0.027 respectively) increased compared with K562 cells transfected with empty vector (p27 kip1:0.159±0.023;p21WAF1:0.188±0.032) and untransfected group (p27Kip1:0.152±0.022;p21WAF1:0.182±0.034) (P<0.05), but the protein expression of CyclinD1 (0.142±0.019)decreased (P<0.05).7 The effects of wtSHIP on apoptosis related proteins in human leukemia cell line K5627.1 After transfection wtSHIP at the third days, the caspase-9 and capsase-3 activity[caspase-9:32.82±5.80nmol/(h·mg); caspase-3: 16.80±1.98nmol/(h·mg)] up-regulated compared with K562/FIV group [caspase-9: 8.75±2.66nmol/(h·mg); caspase-3: 5.48±1.31nmol/(h·mg)] and untransfected K562 cells caspase-9: 8.22±1.25nmol/(h·mg);caspase-3: 5.27±0.58nmol/(h·mg)] (P<0.05).7.2 The bcl-xL protein expression level in K562/wtSHIP group was lower than K562/FIV group and untransfected K562 (K562/wtSHIP: 0.109±0.015; K562/FIV: 0.385±0.043; untransfected group: 0.392±0.049)(P<0.05). The protein expression levels of Bad in K562/wtSHIP was significantly higher than that of K562/FIV and untransfected K562, but no significant difference of protein expression level of Bad was observed among K562/FIV and untransfected group(K562/wtSHIP: 1.018±0.216; K562/FIV: 0.315±0.052; untransfected group: 0.304±0.037). No significant difference of protein expression levels of bax and bcl-2 was observed among K562/wtSHIP,K562/FIV and untransfected K562.7.3 In untransfected group, FIV-GFP group, and wtSHIP-GFP group the protein expression of NF-κB (p65) was significantly down-regulated than that in K562/FIV and untransfected group(K562/wtSHIP:0.483±0.027;K562/FIV组:0.924±0.081;untransfected group:0.937±0.075)(P<0.05); and no significant difference of IκBаprotein level was observed among K562/wtSHIP,K562/FIV and untransfected K562.Conclusions1 This part of study indicated that over expression wild-type SHIP gene could remarkably suppress proliferation of K562 cells, and induce K562 cell apoptosis.2 The effects of wild type SHIP gene on K562 probably via inhibiting the phosphorylation of Akt, and regulating its downstream target protein such as apoptosis related family expression such as Bcl-2 family, Caspase family and the cell cycle related melbourne expression.Part two: Contruction of a lentiviral vector carrying human SHIP mutated gene and its expression in K562 cell lineObjective To construct lentiviral vector carrying human SHIP gene and investigate its expression in K562 cell line. Method Full-length cDNAs of wild type SHIP gene (wt-SHIP) was amplified from the human chondrocytes by RT-PCR, and site-directed mutagenesis was used to obtain full-length cDNAs of the mutated SHIP gene (mut28P/L). The products was cloned into p-Receiver-LV31vector and their sequences was analysed, then subcloned into lentivirus vector pReceiver-LV31, the inserted fragments were analysed and reconfirmed by restriction endonuclease analysis and sequencing. SHIP-lentivirus gene containing green fluorescent protein gene (SHIP-GFP) or the empty vector (FIV-GFP) was transfected into K562 cells. The green fluorescence protein expression of lentivirus plasmids were observed using laser scanning microscopy after 48 hours of transfection. The mRNA expression levels of wtSHIP, muSHIP gene were detected by FQ-PCR. The protein expression levels of wtSHIP and muSHIP were detected by western blot. .Results1 By restriction endonuclease analysis and sequencing, the sequences of SHIPP28L was consistent with that in literature, and the opening frames were matched with the vector sequence.2 Green fluorescent can be seen in transfected 293T after transfection at 48 h. The virus titer was 4.9×106 pfu/ml.3 Green fluorescence signal was distributed throughout K562 cells transfected with wtSHIP and muSHIP vector.The transfection efficiency was K562/wtSHIP-74.6%,K562/FIV-82.5%,K562/muSHIP-76.3%, respectively after transfected with lentivirus on the second day.4 SHIP mRNA and protein were at the highly expression levels in K562/wtSHIP and K562/muSHIP, but in K562/FIV and untransfected group SHIP mRNA and protein level was extremely low, which shown that is transfected into K562 successfully.Conclusions1 Lentivirus expression vector of SHIP gene's mutant of P28L was successfully constructed by using site-directed mutagenesis of SHIP cDNA.2 It was proved that the sequence of the SHIPP28L mutant was correct DNA sequencing.3 Green fluorescent,FQ-PCR and Western blot can be seen in transfected K562 with muSHIP-GFP and wtSHIP-GFP, which shown that it was transfected into K562 successfully.Part three:The study of mutation of SHIP in molecular pathogenesis of leukemiaObjective There are two categories of mutations or gene rearrangements which play important role in molecular mechanisms of leukemogenesis. One class of mutations or gene rearrangements involving mostly tyrosine kinase gene such as BCR/ABL of chronic myelogeous leukemia (CML) confer a proliferative and/or survival advantage to hematopoietic progenitors, and second class of mutations involving mostly transcription factor associated with hematopoietic cell differentiation such as PML/RAR mutation serve primarily to impair hematopoietic differentiation and subsequent apoptosis of cells. Though FLT3-ITD mutation and PML/RAR in acute promyelocytic leukemia (APL) patients as well as c-KIT mutation and AML1/ETO in AML-M2b patients have been reported, the molecular defect involving two categories of mutation are largely unidentified. In this part we focused on elucidate the role of SHIP mutation in leukemogenesis, its association with biological characters of leukemia and its potential in developing new target therpy.Method The recombined lentivirus plasmids muSHIPP28L was transfected stably into human leukemia cells K562. The cell proliferation, cell life cycle and cell apoptosis of K562/muSHIP cells were determined by MTT, fluorescent staining and flow cytometry. Differentially expressed genes were reconfirmed by Western blot.Results1 The enhanced ability of proliferation and DNA synthesis, and decreased apoptosis rate was observed in K562 cells transfected with muSHIPP28L, and the same changes had been observed in K562/FIV and untransfected K562 cells. Increased G0/G1 cells was observed in K562/wtSHIP cells by flow cytometry result, but no such a difference was observed in K562/muSHIP.2 The early apoptosis rate in K562/muSHIP(P28L) group[(8.4±1.11)%] was significantly lower than that in K562/wtSHIP group[(38.4±3.08)%] by Hoechest stainging, no significant difference of the early apoptosis rate was observed between K562/muSHIP and K562/FIV[(8.06±0.95)%]. The SHIP mutant could not induce K562 cell apoptosis.3 SHIPP28L mutant can't down-regulate phosphorylation of Akt308 and 473(p-Akt308:0.334±0.031;p-Akt473:0.826±0.163).4 The cell cycle related molecular expression levels of p21WAF1 and p27KIP1 in K562 cells transfected with wtSHIP were significantly up-regulated, but the protein expression of cyclin D1 down-regulated; no changes indicated above in K562/muSHIP.5 The activity of caspase-3 and capase-9 in K562/muSHIP group were significantly lower than those in K562/wtSHIP group(P<0.05), no significant difference was seen between K562/FIV group and untranfected group(P>0.05).6 The protein expression of bcl-xL decreased in K562/wtSHIP group, but no changes indicated above in K562/muSHIP(0.367±0.028). The expression of bad protein in K562/muSHIP group (0.322±0.063) was lower than that in K562/wtSHIP group , no changes indicate among K562/muSHIP,K562/FIV group and untransfected group(P>0.05). There was no significant difference of bcl-2 expression was observed among K562/muSHIP group,K562/wtSHIP group,K562/FIV group and untransfected group ( P > 0.05 ) . The expression of bax protein in K562/wtSHIP group is a little higher than that in K562/muSHIP group(0.329±0.025), but no significant difference between them(P>0.05).7 High pressure liquid chromatography result shown that the PI3,4,5-P3 level was decreased in K562/wtSHIP,no changes above indicate above in K562/muSHIP group,K562/FIV group and untransfected group. The PI3,4P2 level in K562/wtSHIP group was significantly higher than that in K562/muSHIP group,K562/FIV group and untransfected group(P<0.01), but no significant difference of PIP3 and PI3,4P2 were seen among the last three group(P>0.05).Conclusions1 Site-directed mutation of SHIP gene did not influence on the expression level of gene protein related to the PI3K/Akt signal pathway in the human leukemia cell line K562, however, also decrease ability of apoptosis of K562.2 There are significant difference of caspase-3,caspase-9 and NFκB activity between K562 cells transfected by wild-type SHIP and SHIPP28L mutant.3 The effects of SHIP gene on the regulation the protein expression in the typical PI3K/Akt signal pathway might be an important molecular mechanism that SHIP gene suppress the proliferation and induce the apoptosis of K562, and it depend on the structure and function of normal.