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.

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.

[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.

[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.

[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.

[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.

[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.

[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.

[Effects of sodium orthovanadate on proliferation and apoptosis in raji cells and its mechanism].

In order to investigate the role and the mechanism of protein tyrosine phosphatase (PTPase) signaling pathway in the regulation of proliferation, cell cycle and apoptosis in lymphoma cells, the effects of sodium orthovanadate, Na(3)VO(4), a specific PTPase inhibitor, were explored on Raji lymphoblast-like cell line by MTT assay and CFU-Raji culture, morphologic observation, DNA gel electrophoresis, FCM and RT-PCR. Results showed that MTT assay and CFU-Raji culture demonstrated that sodium or thovanadate inhibited the growth of Raji cells in a concentration-dependent fashion; morphologic observations showed that Raji cells exhibited cytoplasm shrinkage, cytoplasm membrane blebbing, nuclear fragmentation and chromatin condensation forming crescents along nuclear membrane characteristic of apoptosis in the presence of Na(3)VO(4); DNA gel electrophoresis revealed typical DNA ladder reminiscent of DNA cleavage at internucleosomal sites in Na(3)VO(4) treated cells; FCM and RT-PCR indicated that Na(3)VO(4) intervention increased the fraction of annexin V(+) PI(-) cells, reduced the value of mitochondrial transmembrane potential, induced G(2)/M arrest and down-regulated the expression of Bcl-2 and cyclin B1 at both mRNA and protein level in a concentration-dependent manner. It was concluded that PTPase pathway might be implicated in the regulation of cell proliferation, cell cycle and apoptosis, and PTPase specific inhibitor Na(3)VO(4) could induce Raji cell growth inhibition, G(2)/M arrest and apoptosis via down-regulation of Bcl-2 and cyclin B1, and reduction of mitochondrial transmembrane potential.

[In vitro effects of mevastatin on the proliferation and apoptosis in human multiple myeloma cell line U266].

In order to investigate the anti-tumor activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors and the mechanism underlying the cell proliferation and apoptosis modulated in myeloma cells, the effects of mevastatin, an HMG-CoA reductase inhibitor, on cell growth, cell cycle progression and apoptosis in U266 human multiple myeloma (MM) cell line in vitro were explored by MTT colorimetric assay, morphologic observation, flow cytometry, DNA gel electrophoresis, and RT-PCR. The results demonstrated that mevastatin inhibited the growth of U266 cells in time- and dose-dependent manners. Cell cycle analysis showed that U266 cells underwent G(0)/G(1) arrest under exposure to mevastatin, but it did not affect p27 expression at both mRNA and protein level. Morphologic observations revealed cytoplasm shrinkage, nuclear condensation and fragmentation in mevastatin-treated cells, and fraction of annexin V(+)PI(-) cells increased significantly in the presence of the agent as determined by flow cytometric assay. In addition, mevastatin caused the collapse of mitochondrial transmembrane potential (Deltapsim), induced DNA fragmentation, and down-regulated the mRNA expression of bcl-2. The growth-inhibitory, cell cycle arresting, and proapoptotic effects of mevastatin in U266 cells could be effectively reversed by the addition of mevalonate (MVA), the immediate endproduct of the reaction catalyzed by HMG-CoA reductase. It is concluded that mevastatin suppresses proliferation by inducing G(0)/G(1) phase arrest and triggering apoptosis via down-regulation of bcl-2 and reduction of Deltapsim, which may be attributed to the inhibition of MVA pathway by mevastatin. Statins including mevastatin may find their future application in the treatment of MM.

[Effects of bcl-2 antisense oligodeoxynucleotide on proliferation and apoptosis of Raji cells].

OBJECTIVE: To study the in vitro antitumor activity of bcl-2 fully phosporothioated antisense oligodeoxynucleotide (bcl-2 ASODN) to malignant lymphoblastic cells. METHODS: Proliferation and apoptosis of Raji cells incubated with bcl-2 ASODN were evaluated by MTT assay, flow cytometry (FCM) and electron microscopy, and the level of bcl-2 protein and mRNA expression were assessed by FCM and RT-PCR, respectively. RESULTS: MTT assay demonstrated that bcl-2 ASODN could partially inhibit the growth of Raji cells. After incubated with ASODN for 48 hours, Raji cells exhibited characteristic morphologic changes of apoptosis, including cytoplasm membrane blebbing, chromatin condensation crescents formation and nuclear fragmentation. The apoptosis rate of Raji cells treated with 20 micromol/L bcl-2 ASON for 72 hrs was 43.86% which is significantly higher than that of control (10.05%). The bcl-2 ASODN induced apoptosis of Raji cells was accompanied by declined expression of bcl-2 mRNA, which decreased to 0.88% at 72 hrs and was significantly lower than that of control (79.54%). CONCLUSION: bcl-2 ASODN induced Raji cells apoptosis by downregulating bcl-2 protein.

[Effects of aminophylline on proliferation and apoptosis in Raji lympho-blastoid cell line].

The aim of this study was to investigate whether and how phosphodiesterase (PDE) inhibitors modulate the proliferation, cell cycle and apoptosis in lymphoma cells. The effects of aminophylline (AM), a non-specific PDE inhibitor, on Raji cells were explored in vitro. MTT assay, light and transmission electron microscopy and annexin V staining were used to observe cell proliferation, morphologic changes and apoptosis rate in AM-treated cells, and FCM and RT-PCR techniques were adopted to detect the effect on cell cycle, the expression of cyclin B1 and Bcl-2 and mitochondrial transmembrane potential in AM-treated cells. The results showed that AM inhibited the growth of Raji cells in a concentration-dependent manner. Morphologic observations showed apoptosis changes in AM-treated cells, including cytoplamic shrinkage, cytoplasmic bubbling, karyopyknosis and nuclear fragmentation. FCM and RT-PCR detection showed that AM intervention increased the fraction of annexin V(+) cells, reduced the value of mitochondrial transmembrane potential, induced S phase arrest, and down-regulated the expression of Bcl-2 at both mRNA and protein level and cyclin B1 protein in a concentration-dependent manner. It is concluded that PDE inhibitor aminophylline may induce Raji cell growth inhibition, S phase arrest, apoptosis via down-regulation of Bcl-2 and reduction of mitochondrial transmembrane potential.

mitochondrial ceramidase overexpression up-regulates Bcl-2 protein level in K562 cells, probably through its metabolite sphingosine-1-phosphate.

Recently, a mitochondrial ceramidase has been identified and cloned, whose mitochondrial localization strongly suggests the existence of an unexpected mitochondrial pathway of ceramide metabolism that may play a key role in mitochondrial functions, especially in the regulation of apoptosis. To explore the biological effect of mitochondrial ceramidase on cells, pcDNA 3.1/His-CDase plasmid, containing mitochondrial ceramidase cDNA sequence, was transducted into K562 cells mediated by liposome, and G418 was used to screen for positive colonies. A stable transfected K562 cell line was established and named as 'K562TC'. The difference between K562 and K562TC cells in chemotheraputic cytotoxicity response and serum-withdrawal resistance and Bcl-2 protein expression were evaluated by MTT assay, annexin V/PI test, flow cytometry or Western blotting, respectively. The results showed that although survival was comparable between K562 and K562TC cells after exposed to adriamycin, etoposide or arsenious acid, K562TC cells with elevated Bcl-2 protein expression level as identified by FCM or Western blotting revealed stronger resistance to apoptosis induced by serum withdrawal than their parental cells. Inhibition of mitochondrial ceramidase expression in K562TC cells by its specific antisense oligodeoxynucleotide was correlated with a decrease in Bcl-2 protein level. N, N-dimethylsphingosine, a sphingosine kinase inhibitor, depleted intracellular sphingosine-1-phosphate production, also abrogated Bcl-2 protein expression in K562TC cells, while Bcl-2 protein level in K562 cells was up-regulated by exogenous sphingosine-1-phosphate. It is concluded that mitochondrial ceramidase overexpression in K562 cells leads to markedly elevated level of Bcl-2 protein and results in more resistance to serum withdrawal. This effect is initiated not by sphingosine, the direct metabolite of mitochondrial ceramidase, but via sphingosine-1-phosphate, its phosphorylated form. This is the first evidence that mitochondrial ceramidase, through its sphingoid metabolite sphingosine-1-phosphate, up-regulates Bcl-2 protein expression in K562 cells.