Role of circulating CD14++CD16+ monocytes and VEGF-B186 in formation of collateral circulation in patients with hyperacute AMI.

Introduction: Collateral formation is insufficient in some patients with acute myocardial infarction (AMI). Peripheral blood CD14++CD16+ monocytes (intermediate monocytes; IM) and vascular endothelial growth factors (VEGFs) are associated with formation of collateral circulation. Methods: We enrolled 49 patients with AMI who underwent emergency percutaneous transluminal coronary intervention (PCI) (Group A) and 27 patients underwent delayed PCI 1 week after AMI (Group B). The percentage of circulating IM and levels of VEGFs in circulation were determined on day 8th. Left ventricular ejection fraction (LVEF) was measured 3 months after AMI. Results: The peripheral levels of IM and serum VEGF levels on day 8th were significantly higher in patients with well-developed collateral circulation in Group A than those in Group B. The levels of circulating VEGFs in the collateral circulation (+) subgroup in Group B were lower than those in the collateral circulation (-) subgroup. Moreover, the serum VEGF-B186 levels positively correlated with IM. Conclusions: Hyperacute collateral formation in patients with AMI correlated with a higher percentage of CD14++CD16+ monocytes and VEGF-B186 levels in the circulation, which was associated with milder left ventricular remodeling. The regulation of CD14++CD16+ monocytes and VEGF-B may be critical to the formation of collateral circulation and to healing AMI.

GM-CSF-mediated inducement of bone marrow MDSCs by TSA and effect on survival of graft in mice.

OBJECTIVE: This study analyzed the effect of HDAC inhibitor, trichostatin A (TSA), in inducing granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated bone marrow (BM) cell differentiation to myeloid-derived suppressor cells (MDSCs) in vitro and in vivo. METHODS: BM cell differentiation to CD11b + GR-1 + MDSCs was achieved by in vitro culture with TSA and GM-CSF, and the collected cells were analyzed by mixed lymphocyte culture to identify suppressive actions against effector T cells. RT-PCR and ELISA were conducted to analyze the CCL5 mRNA and protein levels in TSA + GM-CSF + BM, GR-1 + MDSCs and GR-1 + MDSC + CCL5 groups. The survival of cardiac grafts was compared between groups. RESULTS: TSA was beneficial for the GM-CSF-mediated BM differentiation to CD11b + GR-1 + MDSCs. Adoptive transfer of GR-1 + MDSCs was powerful in suppressing CD4 + CD25-T cell proliferation and the effect was mediated by iNOS and HO-1; it also increased CCL5 gradient concentration between grafts and plasma to recruit Treg to grafts and prolong the survival of the grafts. Survival analysis revealed that the survival of grafts after adoptive transfer of GR-1 + MDSCs could be prolonged. CONCLUSION: This study helps in further research on the application value of MDSCs in the field of transplant, and may provide a new thought for the cell therapy in inducing immune tolerance in organ transplant.

The Reparative Effects of Human Adipose-Derived Mesenchymal Stem Cells in the Chemotherapy-Damaged Thymus.

Hematological patients who accept chemotherapy always develop secondary tumor or even die of severe infections. As an important central lymphoid organ, the thymus is frequently damaged during chemotherapy. Previous studies showed that the mesenchymal stem cells (MSCs) can promote the proliferation and repair of epithelial cells in thymus. The purpose of our study is to investigate the reparative effects of human adipose-derived mesenchymal stem cells (hADMSCs) in chemotherapy-treated damaged thymus. Eighty mice were randomly divided into four groups: normal group, model control group, hADMSCs untreated group, and hADMSCs treated group. The mice were injected intraperitoneally with dexamethasone sodium phosphate (Dex 20 mg/kg), except the normal group. Then, the chemotherapy models were obtained after 1 week; the treated group was infused intraperitoneally with hADMSCs, whereas the model control group was injected with equal volumes of normal saline. The hADMSC's infusion day was regarded as day 0. The mice were sacrificed at different time points (days 3, 7, 10, and 14). The pathological structure and the function of the thymus, the recovery of T-lymphocyte subpopulation, and the proportion of regulatory T (Treg) cells in spleen and peripheral blood were detected. Additionally, we transfected hADMSCs by lentivirus with green fluorescent protein (GFP) to confirm whether they home to thymus and detected the expressions of cytokines that are associated with the development of thymus in hADMSCs and thymus. The results of the study showed that the hADMSCs treated group had a more rapid recovery in terms of thymic pathological structure and function. The hADMSCs could home to the damaged thymus and secrete cytokines that played important roles in repairing damaged thymus. The results indicated that hADMSCs could repair the damaged thymus caused by chemotherapy and improve the immune microenvironment, which may be a potential treatment for hematological patients.

Impacts of K562 cells towards activities of Toll-like receptor pathway of human mesenchymal stem cell-bone marrow.

The study aim was to investigate the impacts of K562 cells towards the activities of Toll-like receptor pathway of human mesenchymal stem cell-bone marrow (HMSC-bm). The in vitro co-culture of HMSC-bm and K562 cells was set as the experiment group (HMSC-bm + K562), the HMSC-bm cultured alone was set as the control group (HMSC-bm), the expressions of six interested genes and their proteins, namely MyD88, P38, NF-κB, TAB1, TLR3 and TBK1, of the Toll -like receptor signaling pathway were detected and compared, as well as the secretions of such cytokines as IL-6, IL-8, TNF-α and IFN-α in the cell supernatant, which were regulated by the Toll-like receptor pathway. The expressions of MyD88, P38, TAB1 and TLR3 of the HMSC-bm + K562 group were higher than the HMSC-bm group, while that of TBK1 was lower, and the NF-κB expression showed no significant difference between the two groups (P > 0.05). Compared with the HMSC-bm group, the supernatant of HMSC-bm + K562 group exhibited the higher secretion levels of IL-6 and IL-8, while that of IFN-α was just contrary, and the differences were significant (P < 0.05). The secretion levels of TNF-α within the two groups were not significantly different (P > 0.05). The co-culture of K562 and HMSC-bm could induce the activity changes of Toll-like receptor pathway of HMSC-bm, which was beneficial towards the proliferation of K562 cells.

Correlation between osteocalcin-positive endothelial progenitor cells and spotty calcification in patients with coronary artery disease.

Immature endothelial progenitor cells (EPC) carrying osteocalcin (OCN) might mediate vascUlar calcification in coronary artery disease (CAD). Spotty calcification within atherosclerotic plaque is associated with cardiovascular events. The aim of the present study was to assess the correlation between immature EPC levels and spotty calcification in CAD patients. In the 224 CAD patients studied, 76 had acute myocardial infarction (AMI), 102 had unstable angina pectoris (UAP), and 46 had stable angina pectoris (SAP). The levels of OCN-positive (OCN+) EPC were analysed by flow cytometry. The status of spotty calcification was determined by cardiac computed tomography angiography. OCN+ EPC and calcium deposits were significantly increased in acute coronary artery syndrome (ACS) when compared with those in SAP patients. Positive correlation was also revealed between the number of OCN+ EPC and the frequency of spotty calcification and levels of serum high-sensitivity C-reactive protein (hs-CRP) and serum alkaline phosphatase in AMI and UAP patients. In summary, the number of OCN+ EPC is positively related to the frequency of spotty calcification in ACS patients. Serum hs-CRP and serum alkaline levels are thought to contribute to the elevation of OCN+ EPC.

Combining serum and urine biomarkers in the early diagnosis of mild cognitive impairment that evolves into Alzheimer's disease in patients with the apolipoprotein E ϵ4 genotype.

Possession of the apolipoprotein E (APOE) ϵ4 genotype is a major predictor of progression to Alzheimer's disease (AD), particularly in patients with mild cognitive impairment (MCI). However, the use of APOE genotyping in the diagnosis of MCI is limited due to its low sensitivity and specificity, which often results in a high false-positive rate. In this study, we found that there was a significant decrease in serum BDNF and notable increase in urine AD7c-NTP in MCI patients who harbored the APOE ϵ4 allele. Both serum BDNF and urine AD7c-NTP had higher positive predictive values and were more sensitive biomarkers of MCI. Additionally, a testing strategy employing serum BDNF and urine AD7c-NTP revealed increases in sensitivity, positive and negative predictive values, and predictive ability compared with the use of either biomarker alone, suggested that combinatorial detection might have great potential for translation to the clinic.

Effects of peroxisome proliferator-activated receptor γ in simvastatin antiplatelet activity: influences on cAMP and mitogen-activated protein kinases.

Statins are widely used as hypolipidemic drugs, and have beneficial effects in reducing cardiovascular events. In addition, recent studies on the pleiotropic effects of statins (i.e., simvastatin) reveal that these drugs have many additional anti-atherogenic effects, including antiplatelet activity. The mechanisms may be partly related to activation of peroxisome proliferator-activated receptors (PPARs), which are present in human platelets, and whose activation inhibits platelet aggregation. However, the details of the signaling pathway by which simvastatin inhibits platelet activation via PPARs have not yet been completely established. The aim of this study was to examine the mechanisms by which the PPAR-mediated pathways contribute to the antiplatelet activity of simvastatin. Simvastatin (3-50 µM) induced PPARα and PPARγ activation in a dose-dependent manner in washed platelets. Additionally, simvastatin inhibited collagen-induced platelet aggregation, expression of CD62 and PAC-1, and Ca(2+) mobilization. These effects of simvastatin on platelet responses were strongly reduced by adding a selective PPARγ antagonist (GW9662), but not PPARα antagonist (GW6471). Moreover, in the presence of GW9662, simvastatin-mediated increase of cyclic adenosine monophosphate (cAMP) production, vasodilator-stimulated phosphoprotein (VASP) Ser(157) phosphorylation and inhibition of Akt phosphorylation were markedly reversed. Furthermore, simvastatin was found to inhibit phosphorylation of mitogen-activated protein kinases (MAPKs, i.e., p38 MAPK, ERK) by increasing the association between PPARγ and the components of MAPKs after platelet activation. Taken together, the present results confirm that simvastatin inhibition of platelet activation is mediated by PPARγ-dependent processes, which involves mediating MAPKs signaling, increase of cAMP formation and VASP Ser(157) phosphorylation, inhibition of Akt phosphorylation and intracellular Ca(2+) mobilization.

Artesunate induces apoptosis through caspase-dependent and -independent mitochondrial pathways in human myelodysplastic syndrome SKM-1 cells.

Artesunate (ART) is a semi-synthetic derivative of artemisinin extracted from Artemisia annua (sweet wormwood) that is conventionally used in anti-malarial drugs and more recently in medications that induce tumor cell apoptosis. Here, we investigated the effects and mechanistic pathways of ART in human myelodysplastic syndrome (MDS), a condition that commonly progresses to acute myeloid leukemia (AML). Human MDS SKM-1 cells, primary bone marrow (PBM) mononuclear cells from patients with refractory anemia with excess blasts (RAEB) or MDS-AML (MDS cell group), and PBM stromal cells from three patients without hematological diseases (non-MDS cell group) were cultured for 24, 48, or 72 h with or without various ART concentrations. CCK-8, western blot, JC-1 fluorescence, and Annexin-V/Propidium iodide (PI) labeling were used to assess cell proliferation, protein levels, mitochondrial membrane potentials (MMPs) and apoptosis, respectively. ART administration dose- and time-dependently inhibited SKM-1 proliferation. At 24, 48, and 72 h, ART IC50 values were 89.92, 4.24, and 1.28 µmol/L, respectively. ART only significantly inhibited proliferation in the MDS cell group, but it has little impact on proliferation of non-MDS cells. ART decreased MMPs, and dose-dependently induced SKM-1 cell apoptosis, peaking at 82.9% when treated with 200 µmol/L ART for 24h. Caspase-3 and -9 activation, poly(ADP-ribose) polymerase cleavage, decreased Bcl-2/Bax ratio and apoptosis inducing factor nuclear localization were implicated in apoptosis. Our results indicate that ART effectively induces apoptosis in SKM-1 cells through both caspase-dependent and -independent mitochondrial pathways.

Methylation and decreased expression of SHP-1 are related to disease progression in chronic myelogenous leukemia.

Despite the unprecedented success of tyrosine kinase inhibitors (TKIs) in treating chronic myelogenous leukemia (CML), some patients nevertheless progress to advanced stages of the disease. Thus far, the biological basis leading to CML progression remains poorly understood. SH2-containing tyrosine phosphatase 1 (SHP-1) is reported to bind to p210BCR­ABL1 and to function as a tumor suppressor. Furthermore, its substrates have been found to be essential for p210BCR-ABL1 leukemogenesis or CML progression. In the present study, we found that SHP-1 mRNA and protein levels were markedly decreased in patients in the accelerated and blastic phases of CML (AP-CML and BP-CML) compared to those in the chronic phase (CP-CML). In vitro, we demonstrated that overexpression of SHP-1 reduced p210BCR-ABL1 protein expression and activity in the K562 CML cell line and negatively regulated the AKT, MAPK, MYC and JAK2/STAT5 signaling pathways. Moreover, using a methylation-specific polymerase chain reaction (MSP) assay, abnormal methylation of the SHP-1 gene promoter region was found both in K562 cells and bone marrow (BM) or peripheral blood (PB) cells from AP-CML and BP-CML patients. In conclusion, our findings suggest that decreased expression levels of SHP-1 caused by aberrant promoter hypermethylation may play a key role in the progression of CML by dysregulating BCR-ABL1, AKT, MAPK, MYC and JAK2/STAT5 signaling.

Combining trastuzumab and cetuximab combats trastuzumab-resistant gastric cancer by effective inhibition of EGFR/ErbB2 heterodimerization and signaling.

The anti-ErbB2 antibody trastuzumab has currently been approved for ErbB2-positive gastric cancer. Despite the effectiveness of trastuzumab, resistance is common. Thus, there is an urgent need to overcome trastuzumab resistance. Here, we obtain a trastuzumab-resistant cell line, which is derived from the human gastric cancer NCI-N87 cell line, by modeling the development of acquired resistance in patients. Our data show that combining trastuzumab and cetuximab leads to a significant decrease in EGFR/ErbB2 heterodimers and signaling compared with either antibody alone, and the combination results in greater antitumor activity against the trastuzumab-resistant NCI-N87 cell line, both in vitro and in vivo, suggesting that a combined EGFR/ErbB2 inhibition may overcome trastuzumab resistance.

Effect of bone marrow mesenchymal stem cells from blastic phase chronic myelogenous leukemia on the growth and apoptosis of leukemia cells.

Chronic myelogenous leukemia (CML) has a typical progressive course with transition from a chronic phase to a terminal blast crisis phase. However, the mechanisms that lead to disease progression remain unclear. Bone marrow mesenchymal stem cells (BMMSCs) play important roles in maintaining the bone marrow microenvironment. In the present study, the biological characteristics of BMMSCs were determined including proliferation, apoptosis and secretion of cytokines during blastic phase CML (CML-Bp). The effect of BMMSCs in CML-Bp on K562 human CML cells and the CML-Bp original generation leukemia cells were also explored. Our results showed that CML-Bp BMMSCs protect tumor cells and increase their anti-apoptotic ability through regulating the expression of apoptosis-related proteins and activating the Wnt pathway.

[Effect of SHIP mutation on invasion and migration of K562 leukemia cells].

OBJECTIVE: To explore the effect of mutation in PxxP domain of SHIP on migration and invasion of leukemia cells and its mechanism. METHODS: The lentiviral vector mediated wild type SHIP (wtSHIP) and mutant SHIP (muSHIP) plasmids were transfected into K562 cells through gene transfection techniques. Expression of SHIP at mRNA and protein level was detected by real-time PCR and Western blot, respectively. Transwell assay was used to analyze the difference between the migration and invasion ability of the K562/wtSHIP and the K562/muSHIP cells after transfection. Primary migration associated factor FAK, MMP and NF-κB were assayed by Western blot. RESULTS: After transfection, the SHIP expression in transfected K562 cells were significantly increased. Compared with the migration ability of K562/wtSHIP\[(15.8 ± 1.4)%\], that of K562/muSHIP cells \[(54.3 ± 2.4)% \] increased greatly and almost at the same level of that of K562/pFIV\[(50.3 ± 3.8)%\] (P < 0.01). The invasion assay also showed that K562/wtSHIP\[(32 ± 6)/HP\] has a lower invasion ability than that of the K562/muSHIP group \[(83 ± 16)/HP\] and K562/pFIV group \[(78 ± 13)/HP\] (P < 0.01). Western blot analysis showed that the expression of p-FAK and NF-κB was up-regulated in K562/muSHIP group compared to that of the K562/wtSHIP group. CONCLUSIONS: The results confirmed that mutation in PxxP domain of SHIP gene played an important role in negative regulating function of SHIP gene. The mutation affects the cell migration and invasion ability through increase in MMP-9 expression, FAK phosphorylation and NF-κB activation. It suggested that the mutation of PxxP domain in SHIP gene might be pathogenic, and be one of the reasons for SHIP abnormality in leukemia.

Rapamycin provides a therapeutic option through inhibition of mTOR signaling in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a neoplasm of myeloid progenitor cells expressing Bcr-Abl fusion protein. However, some patients with CML are less likely to respond to imatinib, the inhibitor of Bcr-Abl kinase. Recent studies showed that mTOR pathway can increase responses to imatinib. The analysis of mTOR pathway in CML may provide new insights into possible targets of novel therapies. Therefore, we examined the expression of mTOR pathway molecules in bone marrow cells from CML patients and effect of rapamycin on K562 cells in vitro. Western blot analysis showed the visibly higher phosphorylation of mTOR (70.6%), 4E-BP1 (76.5%) and p70S6K (73.5%) in bone marrow cells from CML patients. Moreover, treatment of CML cell line (K562) with rapamycin resulted in a decrease of phosphorylation of mTOR, 4E-BP1 and p70S6K. In vitro, the cell viability in groups with rapamycin treatment displayed a significant decrease in a dose-dependent manner by MTT. The data presented an increase of G0/G1 phase cells and decrease of S phase cells after rapamycin treatment, and the decreased expression of cyclinD1, higher expression of p21 at mRNA level was also detected in K562 with rapamycin. Treatment with 20 nmol/l or more rapamycin could increase apoptotic cells, decrease expression of bcl-2 and activate caspase-3. In conclusion, the mTOR pathway might be involved in chronic myelogenous leukemia. Inhibition of mTOR pathway could interfer with cell proliferation and increase cell apoptosis in K562 cells. It suggested that mTOR might be an important therapeutic target for myelogenous leukemia.

Effect of interaction of magnetic nanoparticles of Fe3O4 and artesunate on apoptosis of K562 cells.

The present study evaluated whether the magnetic nanoparticles of Fe(3)O(4) (MNPs-Fe(3)O(4)) could enhance the activity of artesunate (ART), and to explore its potential mechanisms. Cytotoxicity of the copolymer of ART with MNPs-Fe(3)O(4) on K562 cells was detected by MTT assay and the apoptosis rate of K562 cells was measured by flow cytometry. Protein expression levels of bcl-2, bax, bcl-rambo, caspase-3, and survivin in K562 cells were measured by Western blot. After being incubated with the copolymer of ART with MNPs-Fe(3)O(4) for 48 hours, the growth inhibition rate of K562 cells was significantly increased compared with that of K562 cells treated with ART alone (P < 0.05), and the apoptosis rate of K562 cells was increased significantly compared with that of K562 cells treated with ART alone, suggesting that MNPs-Fe(3)O(4) can enhance the activity of ART. Interestingly, the copolymer-induced cell death was attenuated by caspase inhibitor Z-VAD-FMK. Our results also showed that treatment with the copolymer of MNPs-Fe(3)O(4) and ART increased the expression of bcl-2, bax, bcl-rambo, and caspase-3 proteins, and decreased the expression of survivin protein in K562 cells compared with ART treatment alone. These results suggest that MNPs-Fe(3)O(4) can enhance ART-induced apoptosis, which may be related to the upregulation of bcl-rambo and downregulation of survivin.

[Preliminary study on the effect of TRAIL on adhesion and apoptosis of multiple myeloma cell line RPMI8226 and its mechanism].

The present study was purposed to investigate the inhibition effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) on growth of RPMI8226 cells and adhesion between RPMI8226 cells and bone marrow stroma cells (BMSC), and to explore its mechanism as well. The inhibition effects of TRAIL on cells growth and adhesion were assayed by MTT; cell apoptosis was detected by Annexin V and PI; expression of genes bax, bcl-2, mcl-1, CARP1, CARP2, XIAP and cFLIP were determined by semi-quantitative RT-PCR; apoptosis-related protein expression was detected by Western blot. The results showed that TRAIL inhibited the proliferation of RPMI8226 cells in dose- and time-dependent manners. TRAIL induced apoptosis in RPMI8226 cells, the expression level of genes bcl-2, mcl-1, CARP1, CARP2, XIAP and cFLIP decreased, while the expression level of Bax increased, but the expression level of caspase-3 and NF-kappaB P65(RelA) proteins decreased. Moreover, TRAIL up-regulated the expression level of adherent molecule CXCR4 in RPMI8226 cells significantly. It is concluded that TRAIL up-regulated the expression level of adherent molecule CXCR4 in RPMI8226 cells significantly, and induced the apoptosis of RPMI8226 cells. Growth inhibition effect of TRAIL on RPMI8226 cells is in dose- and time-dependent manners.

[As2O3 induces demethylation and up-regulates transcription of SHP-1 gene in human lymphoma cell line T2 cells].

OBJECTIVE: To investigate the methylation of CpG island in the SHP-1 gene promoter and its significance in lymphoma. To evaluate the effects of As2O3 on demethylation of SHP-1 in human lymphoma cell line T2 and on proliferation of T2 cells. METHODS: T2 cells were treated with AsO3. Methylation specific PCR was used to detected the status of SHP-1 methylation in newly diagnosed lymphoma tissues and the T2 cells. The mRNA and protein expression of SHP-1 were determined by FQ-PCR and Western blot. The expression of phospha-c-kit was examined by Westren blot. MTT and flow cytometry were used to determine the growth and apoptosis in T2 cells. RESULTS: T2 cells contained completely methylated SHP-1. Furthermore, there was constitutive c-kit phosphorylation. The expression of SHP-1 was recoverd when the cells exposed to AsO3, and concomitant with increasing SHP-1, a parallel down-regulation of phosphorylated c-kit occurred, so that by day 3 phosphorylated c-kit was barely detectable. As2O3 inhibited the cell growth, and the effects were dose- and time-dependent. As2O3 also increased apoptosis rate of T2 cells in a dose- and time-dependent manner, too, and on the 1, 2, 3 d treatment with AsO3 (2.5 micromol/L), the apoptosis rates were 6.12%, 26.53%, 50.90%, respectively. The frequency of methylation in SHP-1 gene promoter in lymphoma tissues was 87.5% (28/32). In the control group, however, 12 specimens of benign lymph node proliferation showed no methylation in CpG island of SHP-1 gene promoter. CONCLUSION: Hypermethylation of SHP-1 gene promoter in lymphoma indicates the inactivation of SHP-1 gene and its possible role in the tumorigenesis of lymphoma. As2O3 can effectively cause demethylation and inhibit the growth of tumor by reactivating the SHP-1 gene transcription. SHP-1 methylation leading to epigenetic activation of c-kit may have a tentative role in the pathogenesis of lymphoma. Therefore, As2O3 is potentially useful in the treatment of lymphoma as a demethylating agent.

[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). 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.

[The mechanism for SHIP gene to induce the apoptosis of human leukemia cell line K562.].

The src homology 2 (SH2)-domain containing inositol-5-phosphatase (SHIP) is another recently identified lipid phosphatase after phosphatase and tensin homology deleted on chromosome ten gene (PTEN). It plays an important role in negatively regulating the proliferation of hematopoietic cells. The relationship between SHIP and the inhibition of tumor proliferation is rarely reported. The purpose of this study is to evaluate the apoptosis induced by SHIP gene in K562 cell line and to explore the involved signaling pathway. The K562 cells were transfected with human SHIP gene by using the lentiviral vector containing SHIP, and the transfection was verified by fluorescent quantitative PCR (FQ-PCR) and Western blot. Then the effects of SHIP protein expression on cell growth and apoptosis were measured. The levels of p-Akt, bcl-2 family, caspase and the activity of NFkappaB were assayed by Western blot and ELISA, respectively. The results are as follows: (1) Human leukemia cell line K562 was SHIP-negative; (2) Transfection with SHIP gene led to the re-expression of SHIP mRNA and protein in K562, as shown by FQ-PCR and Western blot; (3) The expression of SHIP protein inhibited cell growth and significantly increased apoptosis in K562 cells; (4) Compared to that in control group, the expression level of p-Akt-308 and p-Akt-473 in SHIP-expressing cell group decreased significantly (P<0.01); SHIP activated caspase-9, caspase-3, up-regulated protein levels of bad, p27, down-regulated expression of bcl-xL, while it had no effect on the expression of bcl-2 and bax. Furthermore, the inhibition of NF-kappaB was achieved along with the inactivation of Akt. These data suggest that SHIP gene has potential abilities to inhibit K562 leukemic cell proliferation and induce its apoptosis via inactivating PI3K/Akt pathway. The loss of SHIP might be the explanation of aberrant high-level p-Akt in human leukemia. It may be at least one of the mechanisms by which the loss of SHIP expression contributes to leukemia progression.

[Apoptosis of the adriamycin-resistant leukemia cell line induced by the recombinant mutant human TNF-related apoptosis-inducing ligand combined with arsenic trioxide].

This study was aimed to investigate the effect of recombinant mutant human TNF-related apoptosis-inducing ligand (rmhTRAIL) combined with As(2)O(3) on inducing apoptosis of adriamycin-resistant leukemia cell line K562/A02 (mdr-1(+)). The morphologic changes of cells treated with rmhTRAIL were observed by inverted microscope, taking adriamycin-sensitive cell line K562 (mdr-1(-)) as control; the inhibitory rate of cell proliferation after being treated with rmhTRAIL, As(2)O(3) alone or combined was assayed by MTT method; the apoptosis peaks of K562/AO2 and K562 were quantitatively detected by flow cytometry with PI staining after being treated with rmhTRAIL, As(2)O(3) alone or in combination. The results indicated that the inhibition effect of rmhTRAIL and As(2)O(3) in combination on K562/AO2 and K562 cells was higher than that of riTRAIL and As(2)O(3) alone (p < 0.01), rmhTRAIL combined with As(2)O(3) had synergistic effect in killing K562/AO2 and K562 cells by king's formula. The apoptosis rates of K562/AO2 and K562 cells were 34.93 +/- 0.10% and 10.53 +/- 0.16% (p < 0.01), as well as 5.95 +/- 0.07%, and 3.50 +/- 0.01% (p < 0.05), 50.95 +/- 0.91% and 20.75 +/- 0.95% (p < 0.05) respectively when their cells were treated by rmhTRAIL and As(2)O(3) alone. The apoptosis rate in K562/AO2 group was higher than that in K562 group. It is concluded that rmhTRAIL can induce K562/A02 and K562 cell apoptosis; rmhTRAIL combined As(2)O(3) had synergistic effects; the efficacy of on rmhTRAIL or As(2)O(3) inducing K562/AO2 cell apoptosis is higher than that on their parental cell line K562.

Comparative study of expressions of cytoplasmic CD79a and other B-lymphoid immunomarkers in acute leukemic cells.

To evaluate the expression of cytoplasmic CD79a (CyCD79a) and other commonly used B-lymphoid immunomarkers including cytoplasmic CD22 (CyCD22), CD19, CD20 and CD10 in various acute leukemia cells and to define the most sensitive and specific markers in the diagnosis of precursor B-cell acute lymphoblastic leukemia (pB-ALL), the immunophenotypic data from 221 de novo adult and pediatric acute leukemia patients as studied using multi-parameter flow cytometry in addition to routine morphologic and enzyme cytochemical assay, were retrospectively analyzed. Cytogenetic and/or molecular biological data in all 45 cases of acute promyelocytic leukemia (APL) and 13 cases of acute leukemia suspected as AML with the fusion genes such as AML1/ETO and CBFbeta/MYH11 were investigated. The results showed that CyCD79a and CyCD22 were the most sensitive and specific markers respectively for pB-ALL. Expression of CyCD79a was seen in 100% of 58 cases of pB-ALL. At the same time, none (0%) of all 147 cases of acute myeloid leukemia (AML) and 15 cases of precursor T-cell acute leukemia (pT-ALL) was positive for CyCD22. The conclusion is made that united detection of CyCD79a and CyCD22 is the optimal immune combination for the diagnosis pB-ALL and the distinguishing pB-ALL with AML and pT-ALL.