Effects Of Mesylate Imatinib On The Expression Of SHIP Gene And Apoptosis Of K562 Cells

Objective: The SH2-containing inositol 5′-phosphatase (SHIP) is widely expressed in hematopoietic cells. It is a member of the inositol 5′-phosphatase family. The human SHIP gene is localizated on chromosome 2q36-37.1. SHIP dephosphrylate 3′-phosphorylated PtdIns on the 5′position, decreasing intracellular levels of PtdIns 3,4,5-P3 and PtdIns 1,3,4,5-P4. SHIP selectively hydrolyzes the 5′-phosphate from inositol 1,3,4,5-tetraphosphate and phosphatidylinositol 3,4,5-trisphosphate(PI-3,4,5-P3), two inositol polyphosphates are the products of PI3K which play important roles in maintaining cell survival. PI3K and phospholipids-regulated signaling have been implicated in many cellular functions including cell adhesion, cytoskeletal reorganization, proliferation and cell survival. In particular, PI3K and phospholipids-dependent activation of the serine/threonine protein kinase B(PKB,also known as AKT) have been shown to rescue cells from apoptosis and therefore may have a role in tumorigenesis. These data show that SHIP has a pivotal role in down-regulating PKB activity and is a crucial negative regulator of cell survival and homeostasis.To investigate the function of SHIP in vivo, Liu et al generated SHIP-deficient mice and show that mice lacking SHIP display dramatic hyperplasia of myeloid cells in the bone marrow and spleen, and myeloid infiltration in various organs. In a series of bcr/abl-transformed cell lines, Philadelphia chromosome (Ph)-posotive cell lines, and primary cells from patients with CML, the expression of SHIP was found to be absent or substantially reduced compared to untransformed cell lines or leukemia cells lacking bcr/abl. Sattler et al demonstrated that the expression of SHIP was directly inhibited by bcr/abl. The reduction of SHIP due to bcr/abl was likely to directly contribute to the pathogenesis of CML. To evaluate the possible tumor suppressor role of the SHIP gene in leukemogenesis, Luo et al examined primary leukemia cells from 32 acute myeloid leukemia(AML) and 9 acute lymphoblastic leukemia(ALL) patients. Mutations of SHIP gene were detected in 22% AML patients and 12% ALL patients. 78% of these mutations were lying within the phosphatase active site and other crucial domain of cellular function. No mutations of that was detected in normal control group. At present, it is not clear that the expression of SHIP gene in leukemic cells and the effects of SHIP gene to AKT phosphorylation levels and the evidence for a positive role of SHIP in the bcr/abl positive hematopoietic cells and the signal transduction pathways of SHIP gene on apoptosis in leukemic cells. So the major research contents of this subject were as follows: 1.Real time quantitative polymerase chain reaction was used to detect the expression of SHIP gene in leukemic cells from AL patients and various leukemia cell lines, the clinical significance of the SHIP gene expression in AL patients were explored simutanously; 2.The SHIP gene expression levels were up- regulated in K562 (a bcr/abl positive cell line) cells after treated with mesylate imatinib (MI) (a inhibitor of bcr/abl), so the negative regulator effects of bcr/abl fusion gene to SHIP were preliminary confirmed; 3.Flow cytometry(FCM) was used to analyse the correlation between the level of SHIP gene expression and the level of AKT phosphorylation; 4.Reverse transcript polymerase chain reaction was used to check the effects of SHIP gene on the expression levels of Bcl-2,Caspase-1,Casepase-3 and Casepase-9 genes; The cell proliferation was measured by MTT assay; The cell apoptosis was analyzed by AnnexinⅤ/PI double staining flow cytometry.Methods1 Suspended cell culture and cell treated with mesylate imatinibJurkat cells, Nalm-6 cells, K562 cells, U937 cells, KG-1 cells, HL-60 cells and NB4 cells were cultured in RPMI1640 medium supplemented with 15%(V/V) heat-inactivated fetal bovine serum(Hyclone), 100U/ml penicillin G and 100μg/ml streptomycin sulfate at 37℃with 5% CO2 enrichment. Logarithmically growing cells with viability≥95% as assayed by trypan blue exclusion staining were used for the studies described below. Mesylate imatinib was dissolutioned in high pressure sterilization tri-distilled water and dispersioned in EP tubes at 4℃. The logarithmically growing K562 cells were cultured with mesylate imatinib at different concentrations (0.5μmol/L,1.0μmol/L,5.0μmol/L and 10.0μmol/L). The cells were collected at different time points (3h,6h,12h and 18h).2 The expression of SHIP gene were measured by real time quantitative polymerase chain reaction(RQ-PCR)Total RNA was extracted by using Trizol RNA extraction kit. M-MLV was used for reverse transcriptional synthesis of cDNAs in a total volume 20.0μl reaction system(5×buffer 4.0μl, 5'primer 0.4μg, 3'primer 0.4μg, dNTPs 0.2μg, M-MLV 1.0μg, DEPC 9.0μl, RNA 1.0μg) at 37℃for 1 hour and 95℃for 3 minutes. The PCR protocol for positive model and SHIP gene were as follows: 5×buffer 10.0μl, 5'primer 1.0μg, 3'primer 1.0μg, dNTPs 0.5μg, fluorescent primer 1.0μl, Tag DNA polymerase 1.5μg, cDNA model 5.0μl and ddH2O 30.0μl consisted a total reaction system in 50.0μl. The PCR reaction condition was 93℃for 2 minutes, 93℃for 45 seconds , 55℃for 45 seconds of 1 cycle and total for 40 cycles.3 The levels of P-Akt were checked by Flow Cytometry(FCM )A. Solutions and Reagents: A1 1×phosphate buffered saline(PBS): dissolve 8g Nacl, 0.2 Kcl, 1.44gNa2HPO4 and 0.24g KH2PO4 in 800ml distilled water (dH2O). Adjust the PH to 7.4 with HCL and the volume to 1 liter. Store at room temperature. A2 Formaldehyde(methanol free). A3 Incubation Buffer: dissolve 0.5g bovine serum albumin (BSA) in 100ml 1×PBS, store at 4℃. B. Fixation: B1 Collected cells by centrifugation and aspirate supernatant; B2 Resuspend cells briefly in 0.5-1 ml PBS, then add formaldehyde so that the final concentration is 1%-2% formaldehyde; B3 Fix for 10 minutes at 37℃; B4 Chill tubes on ice for 1 minute. C. Permeabilization: C1 Permeabilize cells by adding ice-cold 100% methnol slowly to pre-chilled cells while gently vortexing so that final concentration is 90% methanol. Alternatively to remove fix prior to permeabilization, pellet cells by centrifugation and resuspend in 90% methanol. C2 Incubate 30 minutes on ice or at 4℃. C3 Proceed with staining or store cells at -20℃in 90% methanol. D. Staining: D1 Count cells using hemacytometer or alternative method. D2 A liquot 5×105 cells into each assay tube(by volume). D3 Add 2-3ml incubation buffer to each tube and rinse by centrifugation. D4 Resuspend cells in 90μl incubation buffer per assay tube. D5 Let cells block in incubation buffer for 10 minutes at room temperature. D6 Add 10μl of conjugated antibody to the assay tubes. D7 Incubate for 30-60 minutes, in the dark at room temperature. D8 Rinse as before in incubation buffer by centrifugation. D9 Resuspend cells in 0.5ml PBS and analyze on flow cytometry.4 Analysis of apoptosis associated genes mRNA expression by RT-PCR methodThe cultured K562 cells were harvested from both control group and MI-treated groups and washed by PBS. Total RNA was extracted by using Trizol RNA extraction kit. M-MLV was used for reverse transcriptional synthesis of cDNAs. Oligonucleotide primer pairs are: 5'-CGA CGA CTT CTC CCG CCG CTA CCG C-3'and 5'-CCG CAT GCT GGG GCC GTA CAG TTC C-3'for bcl-2(318bp); 5'-ATC CGT TCC ATG GGT GAA GGT ACA-3'and 5'-CAA ATG CCT CCA GCT CTG TAA TCA-3'for Caspase-1(600bp); 5'-TTC AGA GGG GAT CGT TGT AGA AGT C-3' and 5'-CAA GCT TGT CGG CAT ACT GTT TCA G-3'for Caspase-3(264bp); 5'-ATG GAC GAA GCG GAT CGG CGG CTC C-3'and 5'-GCA CCA CTG GGG GTA AGG TTT TCT AG-3'for Caspase-9(330bp); 5'-CGA TGC TGG CGC TGA GTA C-3' and 5'-CGT TCA GCT CAG GGA TGA C-3'for GAPDH(450bp). 10μl PCR products were separated in 1.5% agarose gel electrophoresis with 90V for 60 minutes and visualized by goldview staining. 5 The cell apoptosis was analyzed by AnnexinⅤ/PI double staining FCM 1ml K562 cells in each group (1×106/ml) were collected, centrifuged at 4℃for 10 minutes and washed twice with PBS. Resuspended cells in 200μl Binding Buffer. 10μl AnnexinⅤ-FITC and 5μl PI were added in order, according to the manufacture's instruction. Each tube was mixed-up thoroughly and placed in dark for 15 minutes, then 300μl Binding Buffer was added. The percentage of apoptotic K562 cells were measured by FCM within 1 hour.6 The percentage of K562 cells proliferation inhibition was measured by MTT assayLogarithmically growing cells both from control group and MI-treated groups were cultured triplicate in 96-well culture plates. The density of cell was 5×104/ml and 200μl in each well. 4 hours before the ending of culturing, cells in each well were added 10μl MTT solution(10mg/ml). After 4 hours incubation, cells were centrifuged and resuspended with 200μl dimethylsulfoxide(DMSO) to solve the crystallisate completely. The OD value was measured at 570nm with Microplate Reader, and the cell growth curve was drawn and then the proliferation inhibition percentage was calculated.7 The morphological change of K562 cells after it was treated with MI was observed by Wright-Giemsa stainingEach group of K562 cells treated with MI was collected and centrifuged, smears were made and staining by Wright-Giemsa method. The morphological changes of typical apoptotic cells were observed: nuclear contracted, nuclear marginal aggregation, nuclear rupture, membrane process and apoptotic bodies.Results1 The expression levels of SHIP gene in leukemic cells1.1 The expression levels of SHIP gene in leukemic cell linesThe expression level of SHIP gene in Jurkat cells was (6.85±0.25)×106/μgRNA; in Nalm-6 cells was (6.12±0.47)×103/μgRNA; in U937 cells was (3.02±0.23)×105/μgRNA; in KG-1 cells was (2.90±0.39)×105/μgRNA; in HL-60 cells was (8.15±0.69)×103/μgRNA; in NB4 cells was (8.77±0.67)×104/μgRNA; in K562 cells was (3.64±0.79)×102/μgRNA; There was no statistical difference between the expression level of SHIP gene in KG-1 cells and that in U937 cells, but there were significant difference among other cell lines. In all examined cell lines, the expression level of SHIP gene in Jurkat cells was highest, that in K562 cells was lowest.1.2 The expression level of SHIP gene in cells from leukemia patients and clinical significanceThe expression levels of SHIP gene in 36 primary acute leukemia(AL) patients and control groups were (6.86±3.44)×103/μgRNA and (4.31±2.92)×104/μgRNA, respectively.(ρ=0.0005). The expression level of SHIP gene in 12 relapsed AL patients was (1.05±0.53) 104/μgRNA. (Compared with control group,ρ=0.0011). The expression level of SHIP gene in 12 CML patients was only (6.16±7.65)×102/μgRNA. (Compared with control group, untreated group and relapsed group,ρ=0.0000). The complete remission(CR) rate of 36 primary AL patients was 88.89%(32/36). According to the expression level of SHIP gene, the 36 primary AL patients were divided into two groups: groupⅠ: the expression levels of SHIP gene in this group were all lower than average level; groupⅡ: the expression levels of SHIP gene in this group were all higher than average level. There was no significance of CR rate between groupⅠ(87.50%) and groupⅡ(90.00%).2 The effects of MI on the expression level of SHIP gene in K562 cells The bcr/abl fusion gene is positive in K562 cells. K562 cells were cultured with MI at different concentrations (0.5μmol/L, 1.0μmol/L, 5.0μmol/L, 10.0μmol/L). The cells were collected at different time points (0h, 3h, 6h, 12h, 18h). The results showed that MI significantly increased the expression of SHIP gene in a time-and dose-dependant manner. The highest level of SHIP gene in K562 cells was at the point of 18h after it was treated with MI in the 1.0μmol/L group.3 The effects of MI on SHIP gene and the expression level of P-Akt in K562 cells3.1 The expression levels of P-Akt in leukemic cell linesThe expression level of P-Akt in Jurkat cells was (48.76±4.01); in Nalm-6 cells was (102.41±7.52); in U937 cells was (170.19±14.04); in KG-1 cells was (119.23±17.71); in HL-60 cells was (237.12±28.97); in NB4 cells was (94.52±10.76); in K562 cells was (97.38±12.72). The expression level of P-Akt in Jurkat cells was lowest; in HL-60 cells was highest(ρ=0.0016). There was no relationship between the expression level of P-Akt and that of SHIP gene(r=-0.5399).3.2 The changes of P-Akt expression level in K562 cells after it was treated with MIThe expression level of P-Akt in K562 cells was gradually decreased by MI in a time-dependent manner. In the 1.0μmol/L group, the expression level of P-Akt was 92.42 before it was treated with MI, and that decreased to 34.16 after it was treated with MI at the point of 18h.3.3 The correlation between the expression level of P-Akt and SHIP gene in K562 cellsIn 0.5μmol/L, 1.0μmol/L and 5.0μmol/L groups, the expression levels of SHIP gene in K562 cells were gradually increased to the highest levels at the point of 18h after it was treated with MI, the P-Akt levels were gradually decreased to the lowest levels simutanously. There were correlations between P-Akt and SHIP(r=0.8527,-0.9275 and -0.9656). In 10.0μmol/L group, there was no correlation between P-Akt and SHIP(r=-0.0430).4 The influence of SHIP gene on the proliferation and apoptosis in K562 cells4.1 The changes of expression levels of Bcl-2, Caspase-1, Caspase-3 and Caspase-9 in K562 cells after it was treated with MIThere were no significant changes of Bcl-2, Caspase-1 and Caspase-3 expression levels in K562 cells after it was treated with MI at different concentrations and at different time points. The Caspase-9 expression levels were increased by MI at 12h and 18h but not at 3h and 6h. The Caspase-9 expression level was highest in 1.0μmol/L concentration group.4.2 The correlation between SHIP and Caspase-9 genes in K562 cellsThe SHIP gene expression levels were increased by MI at 0.5μmol/L, 1.0μmol/L and 5.0μmol/L concentrations in a time-dependent manner. In 10.0μmol/L concentration group, the highest expression level of SHIP was at 6h point. The expression levels of SHIP at 12h and 18h points were lower than that at 6h point. There was linear correlation between the SHIP and Caspase-9 genes(r=0.8582, 0.6050, 0.6547 and 0.6030 at 0.5μmol/L, 1.0μmol/L, 5.0μmol/L and 10.0μmol/L concentration groups, respectively.).The highest expression level of Caspase-9 gene was occurred in 1.0μmol/L group at 18h.4.3 The percentage of apoptotic K562 cells after it was treated with MIThe apoptotic cells in each group was gradually increased in a time-dependent manner after it was treated with MI. There were no statistical different of the percentage of early apoptotic K562 cells among all concentration groups.4.4 MI inhibited the proliferation of K562 cellsThe results illustrated that the absorbance values of K562 cells decreased in a time-dependent manner after it was cultured with MI. But it had no correlation with the concentrations of MI.4.5 The morphological changes of K562 cells after it was treated with MIThe morphological changes of K562 cells was observed under the oil lens by Wright-Giemsa staining. The typical morphological changes of apoptosis were observed: nuclear contracted, nuclear marginal aggregation, nuclear rupture, membrane process and apoptotic bodies.Conclusions1 SHIP gene were expressed in all examined cell lines(T, B and myeloid cell lines), but the expression levels were different. The expression level of SHIP gene in K562 cell line was lowest maybe because of bcr/abl fusion gene inhibited the SHIP expression directly. The expression level of SHIP gene in various subtype groups of leukemia patients were also different. The levels in primary and relapsed groups were lower than that in control group. These results show that the SHIP expression was inhibited in leukemia patients. The expression level of SHIP gene in CML patients was lowest. The bcr/abl fusion gene maybe contribute to this phenomenon.2 The expression level of SHIP gene was significantly increased by MI in K562 cells. This result show that SHIP gene could be up-regulated after bcr/abl fusion gene was inhibited or down-regulated by MI. 3 SHIP gene expression level was up- regulated by MI and P-Akt was down-regulated simutanously. There was negative linear correlation between SHIP and P-Akt. This result show that SHIP has a pivotal role in down-regulating Akt activity and is a crucial negative regulator of cell survival and homeostasis.4 It's the Caspase-9 expression level but not Caspase-1 and Caspase-3 was up-regulated by MI. The expression level of Bcl-2 had no change before and after K562 cells was treated by MI. There was a positive linear correlation between SHIP and Caspase-9. These data show that Caspase-9 maybe involved in the apoptotic pathway caused by SHIP but it should be confirmed via a series of experiments.5 MI can inhibited the proliferation of K562 cells and increased apoptotic cells in a time-dependent manner and it has been confirmed by the morphology changes. These data show that low concentration of MI could cause apoptosis in K562 cells.