Inhibitory effect of 14-3-3ζ on the proliferation of HL-60 cells and HL-60/VCR cells / 中国实验血液学杂志

This study was aimed to investigate the expression and role of 14-3-3ζ in the AML cell lines: sensitive HL-60 and drug-resistant HL-60/VCR cells. Semi-quantitative RT-PCR and Western blot were respectively used to examine the expression of mdr1 mRNA and Pgp in AML cell lines to validate the results of microarray. Western blot was performed to investigate the expression of Pgp, 14-3-3ζ, and anti-apoptosis protein BCL-2, MCL-1 proteins. Immunofluorescence assay was used to detect the subcellular location of 14-3-3ζ protein in HL-60 and HL-60/VCR cells by laser scanning confocal microscopy. Transduction with siRNA was used to silence 14-3-3ζ in AML cell lines. Cell count method and flow cytometry of cell cycle were used to analyze the changes of growth of AML cells. The results found that mdr1 mRNA and Pgp did not expressed in HL-60 cells, but significantly overexpressed in HL-60/VCR cells. Except 14-3-3σ, the expression of other subtypes of 14-3-3 was higher in HL-60/VCR cells than that in HL-60 cells, especially 14-3-3ζ. The higher expression of 14-3-3ζ, BCL-2, MCL-1 protein was observed in HL-60/VCR cells than that in HL-60 cells. These results were same results from gene chip. It was also noticed that 14-3-3ζ was located in the cytoplasma and nuclei of AML cell lines, especially over-expressed in HL-60/VCR cells. Furthermore, suppression of 14-3-3ζ by RNA interference resulted in inhibition of the proliferation of AML cells with decreased protein expression of BCL-2 and MCL-1, especially in HL-60/VCR cells. It is concluded that 14-3-3ζ plays an important role in proliferation of AML cells and associates with BCL-2 and MCL-1 expression. These results suggested that development of therapy targeting 14-3-3ζ may provide novel, effective strategies for refractory and relapsed AML.

Clinical analysis on 28 patients with hemophagocytic lymphohistiocytosis syndrome / 中国实验血液学杂志

In order to profoundly understand the clinical and laboratorial characteristics and inducing factors of hemophagocytic lymphohistiocytosis syndrome (HLH), 28 HLH patients received from 2004 to 2009 years in our hospital were analyzed retrospectively. The results indicated that all of the patients had a history with prolonged fever (more than 1 week), pancytopenia, hepatosplenomegaly, elevated ferritin level, hypofibrinogen, and hemophagocytosis in bone marrow. HLH was the first characteristic sign of malignant lymphoma in 9 patients; 1 patient had a clinical manifestation similar to fulminant hepatic failure; severe psycho-abnormality occurred in 1 HLH patient and pronounced hemophagocytosis were detected in his cerebrospinal fluid; 1 patient was eventually diagnosed as having HLH by the findings in a lymph node biopsy showing obvious hemophagocytosis. Additionally, the analysis of underlying factors in 28 patients with HLH indicated 11 patients with EB virus-associated HLH, 11 with lymphoma-associated HLH, 2 with Leishmania-associated HLH, and 3 with autoimmune disease-associated HLH. It is concluded that HLH disease is characterised with high heterogenicity in both clinical features and inducing factors; in addition, the patients from a pasturing area should be paid attention to parasite infection such as leishmania.

Effects of xbp-1 gene silencing on bortezomib-induced apoptosis in human multiple myeloma cells / 中国实验血液学杂志

This study was purposed to investigate the effect of xbp-1 gene silencing on bortezomib-induced apoptosis in multiple myeloma cell line NCI-H929 (H929). After xbp-1 gene expression was interfered by small hairpin RNA, the cell apoptosis was assayed by flow cytometry with Annexin V-FITC/PI staining, and the expression level of XBP-1 protein was detected by Western blot. The results showed that XBP-1 protein level of H929 cells was inhibited effectively by the PLL3.7 lentiviral vector mediated expression xbp-1 shRNA. The apoptosis rate was significantly higher in xbp-1 shRNA-expressing cells than in untreated control group [(10.13±0.61)% vs (2.5±0.2)%, p<0.05]. After treatment with bortezomib, the apoptosis rate of XBP-1 protein functionally deficient H929 cells was significantly higher than those in vector control group [(45.07±1)% vs (19.53±0.8)%, p<0.05]. It is concluded that xbp-1 gene silencing can significantly enhance the pro-apoptotic activity of bortezomib in multiple myeloma cells.

Bortezomib-induced BiP expression and apoptosis in multiple myeloma cells / 中国实验血液学杂志

This study was aimed to explore the effect of bortezomib on the apoptosis and expression of the molecular chaperone BiP in human multiple myeloma cell line NCI-H929 (H929). After treatment of H929 cells with different concentrations of bortezomib for 24 hours, cell apoptosis was assayed by flow cytometry with Annexin V-FITC/PI staining, and the expression levels of BiP mRNA and protein were detected by RT-PCR and Western blotting analysis. The results showed that bortezomib of different concentrations (20, 40 and 80 nmol/L) induced apoptosis of H929 cells in dose-dependent manner, with apoptotic rates (15.73 +/- 0.67)%, (27.83 +/- 1.26)% and (44.17 +/- 2.25)% respectively, which were significantly higher than that in control (1.21 +/- 0.07%) (p < 0.05). Bortezomib-induced up-regulation of BiP mRNA levels was almost on a parallel with BiP protein when compared with control. Under the similar apoptosis-stimulating conditions with apoptotic rates varying from 40% to 50%, expression levels of BiP mRNA and BiP protein induced by the classical endoplasmic reticulum stressor Brefeldin A (500 ng/ml, 24 h) were almost consistent with those by bortezomib (80 nmol/L, 24 h). It is concluded that bortezomib-induced apoptosis in H929 cells correlates closely with endoplasmic reticulum stress.

PAD regimen for relapsed or refractory patients with multiple myeloma / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the efficacy and safety of PAD [bortezomib (PS-341), doxorubicin and dexamethasone] regimen for relapsed or refractory multiple myeloma (MM).</p><p><b>METHODS</b>Seventeen patients with relapsed or refractory MM received two to four 21-day cycles of PAD: an intravenous bolus of bortezomib (1.3 mg/m2) on days 1, 4, 8, and 11; doxorubicin 10 mg per day on days 1 to 4, and dexamethasone 40 mg on days 1-4. Response was evaluated according to International Myeloma Working Group Criteria (IMWG 2006), toxicity was graded according to NCI CTCAE (common terminology criteria for adverse events) v 3.0.</p><p><b>RESULTS</b>After 2-4 courses of PAD, 14 patients (82.4%) response, including complete response (CR) in 4 (23.5%), very good partial response (VGPR) in 4 (23.5%), partial response (PR) in 6 (35.3%) and stable disease (SD) in 3 (17.6%). Median time to progression was 9.5 months. The median course to response was 1.6 (1-3). All of 5 patients with extramedullary plasmacytoma achieved at least PR after the first cycle therapy; the plasmacytoma disappeared after 1-2 cycles of PAD. The efficacy was independent of other prognostic factors such as beta2-MG. Adverse events included thrombocytopenia in 9 patients (52.9%), leukopenia in 4 (23.5%), peripheral neuropathy in 4 (23.5%), varicella herpes zoster in 3 (17.6%), fatigue in 6 (35.3%) and diarrhea in 2 (11.7%). All of these adverse reactions could be controlled with routine supportive treatment, only one patient died from respiratory failure during his fifth PAD cycle.</p><p><b>CONCLUSIONS</b>PAD regimen should be considered as an appropriate treatment for relapsed or refractory MM, especially for MM with extramedullary plasmacytoma. Its efficacy is independent of traditional prognostic factors. The side effects are usually manageable.</p>

CMV pp65 gene modified dendritic cells activate autologous T cells / 中国实验血液学杂志

Cytomegalovirus (CMV) infection is a dangerous complication in patients with chronic graft versus host disease (cGVHD). CMV-specific immunity depends on the activity of T cells. This study was aimed to investigate the effect of CMV pp65 gene modified dendritic cells (DCs) on activation of autologous T cells. Lentivirus system was utilized to introduce the CMV full-length pp65 gene into mouse DCs; CpG-DNA was used to induce mature DCs; flow cytometry and immunofluorescence were used to determine the expression of antigen and IFNgamma in T lymphocytes. The results showed that the DCs were infected with lentivirus at a multiplicity of infection (MOI) of 50 with optimal infectious efficiency of 30%-40%; mature DCs expressing pp65 gene could stimulate autologous naive T cells to express CD69 specifically; mature DCs expressing PP65 could stimulate autologous CD4+ or CD8+ T cells to produce IFNgamma. It is concluded that CMV pp65-modified and CpG-DNA-induced mature DCs can activate CMV-specific T lymphocytes in vitro.

Tyrosine kinase inhibitor reverses adriamycin resistance mediated by cell adhesion in RPMI8226 cells / 中国实验血液学杂志

To study the effects of tyrosine-kinase inhibitor STI571 on the adhesion of RPMI8226 cells to fibronectin (FN), cell adhesion mediated adriamycin-resistance and the Rac1 mRNA expression, the adhesion of RPMI8226 cells to fibronectin and drug resistance mediated by cell adhesion were determined by means of crystal violet staining and MTT assays respectively, Rac1 mRNA levels in RPMI8226 cells were examined by semi-quantitative RT-PCR. The results showed that STI571 could inhibit the adhesion of RPMI8226 cells to fibronectin. When RPMI8226 cells had been adhered to FN or BSA-coated wells for 1, 6 and 12 hours, the adhesion rates were (43.71 +/- 2.18)%, (55.63 +/- 1.56)%, and (63.42 +/- 2.46)% respectively. After treatment with STI571 20 micromol/L, the adhesion rates decreased to (15.12 +/- 1.04)%, (17.58 +/- 1.32)% and (17.24 +/- 1.59)% respectively (P < 0.05). The experiment revealed that growth of RPMI8226 cells adhered to FN-coated plates had a significant advantage over growth on BSA-coated plates when exposed to adriamycin (Adr) for 1 hour followed by a 24-hour culture period, and the mean IC(50) value for FN-adhered cells was (1.46 +/- 0.04) micromol/L while mean IC(50) value for BSA control was (0.78 +/- 0.03) micromol/L (P < 0.05). Following treatment with 20 micromol/L STI571, the mean IC50 values for FN and BSA adhered cells were (0.81 +/- 0.05) micromol/L, (0.74 +/- 0.02) micromol/L respectively, there was no significant difference between them (P > 0.05). RT-PCR demonstrated that the relative Rac1 mRNA level (Rac1/GAPDH) in RPMI8226 cells was downregulated following being treated with 20 micromol/L STI571. It is concluded that STI571 can inhibit the adhesion of RPMI8226 cells to fibronectin, reverse cell adhesion mediated adriamycin-resistance, and downregulate Rac1 mRNA level.

Rac1 expression and its effects on the cell cycle progression and apoptosis in human acute leukemic cell line HL-60 / 中国实验血液学杂志

The study was aimed to investigate the expression of Rac1 in human acute leukemic cell line HL-60 and effect of Rac1 on cell cycle progression and apoptosis. The mRNA expression of Rac1 in HL-60 cell line and normal human peripheral blood mononuclear cells (PBMNC) were examined by semi-quantitative RT-PCR. After transfection of HL-60 cells with different concentrations of Rac1 antisense oligodeoxynucleotide (ASODN) by means of FuGENE6, the survival, cell cycle, apoptosis of HL-60 cells were observed through MTT assay, FCM test, Wright-Giemsa, acridine orange/ethidium bromide (AO/EB) and Annexin V-FITC/PI staining test respectively. The results showed that Rac1 relative amount in HL-60 was 0.84 +/- 0.13, while it in the normal PBMNC was 0.26 +/- 0.1 (P < 0.01); the expression of Rac1 in HL-60 cells was significantly upregulated. Compared with sense oligodeoxynucleotide (SODN), HL-60 cell viability, after exposure to ASODN at a concentration of 2.0 g/L decreased, (73.7 +/- 5.0)% vs (93.2 +/- 3.0)% (P < 0.01), while the proportion of G(1) cells increased as (52.1 +/- 6.8)% vs (31.6 +/- 4.7)% (P < 0.05), the percentage of Annexin V positive cells increased, (19.2 +/- 2.1)% vs (4.1 +/- 1.7)% (P < 0.01), and HL-60 cells were observed to have formation of apoptotic bodies. The data presented indicate that Rac1 may be involved in regulation of HL-60 cell cycle and apoptosis, promote overproliferation of HL-60 cells and inhibit their apoptosis.

The study on reversing mechanism of multidrug resistance of K562/A02 cell line by curcumin and erythromycin / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of curcumin (Cur) and erythromycin (EM) on multidrug resistance (MDR) reversal of K562/A02 cell line and their mechanism.</p><p><b>METHODS</b>MTT assay was employed to determine the sensitivity of Cur, EM-treated K562/A02 cells to adriamycin (ADM). Flow cytometry was used to measure intracellular mean fluorescence intensity (MFI) of daunorubicin (DNR). P-gp expression was determined by immunohistochemistry. RT-PCR technique was used to examine the mdr1 mRNA level.</p><p><b>RESULTS</b>IC(50) of ADM in K562/A02 cells was decreased when treated with Cur or EM, and the reversal times (RvT) was 4.9, 3.7 respectively. The RvT reached to 11.3 when treated with Cur (2.5 microg/ml) combined with EM (120 microg/ml). The DNR MFI in K562/A02 cells was significantly lower than that in K562 cells (P < 0.01), and was increased significantly when treated with Cur (2.5 microg/ml) or EM (120 microg/ml) (P < 0.05). There was no significant difference between DNR MFI of K562/A02 cells treated with Cur (2.5 microg/ml) or EM (120 microg/ml). Immunohistochemistry showed that P-gp expression was significantly higher in K562/A02 cells than in K562 cells (P < 0.01), and was reduced in K562/A02 cells treated with each (P < 0.01), though being still higher than that in K562 cells (P < 0.01). P-gp expression of K562/A02 cells treated with each drug for 5 days were lower than that for 3 days (P < 0.01), and lowered further when treated with Cur and EM together (P < 0.01). Mdr1 mRNA level in K562/A02 cells was higher than in K562 cells (P < 0.01), and was decreased when treated with each of the drugs (P < 0.01). The mdr1 mRNA level of K562/A02 cells treated with Cur (2.5 microg/ml) plus EM (120 microg/ml) was decreased most significantly than that treated with other group of drugs. After 5 day treatment the mdr1 mRNA level of K562/A02 cells with Cur (2.5 microg/ml) was lower than that with EM 120 microg/ml (P < 0.01).</p><p><b>CONCLUSIONS</b>Either Cur or EM can partly reverse the multidrug resistance of K562/A02 cells and decrease the expression and function of P-gp in a time-dependent way. MDR reversing effect of Cur combined with EM is stronger than that of Cur or EM alone.</p>

Expression of bFGF and VEGF in acute leukemia and its effects on HL-60 cell growth / 中国实验血液学杂志

The study was to investigate the expression levels of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in the serum of patients with acute leukemia and supernatants of leukemia cell lines as well as effects of VEGF-specific antisense oligodeoxynucleotides (ASODN) on the growth of HL-60 cells. Meanwhile the methods to evaluate the VEGF level in the serum of patients with acute leukemia were explored. The levels of bFGF and VEGF in the serum from 32 patients with acute leukemia and 10 healthy subjects and in the supernatants of 5 various human leukemia cell lines were quantified by means of the enzyme-linked immunosorbent assay (ELISA) and were compared. VEGF levels were evaluated not only without standardization but also after standardized by platelet and finally expressed as VEGF/PLT (pg/10(6)). After with different concentrations of VEGF ASODN, HL-60 cell viability was examined with MTT assay and VEGF levels in supernatants were measured with ELISA, respectively. The results showed that bFGF was detected (3 pg/ml) in 14 out of 32 serum samples from patients with acute leukemia, and the positive (37.5%) was significantly higher than that in healthy controls (10%) (P < 0.01). 3 out of 5 supernanant samples obtained from leukemia cell lines demonstrated positive for bFGF as well. There is no difference of the serum VEGF levels between leukemia patients and healthy controls, but the serum VEGF levels in the serum from leukemia patients were significantly higher than those in healthy controls (P < 0.05) after standardization. 4 out of 5 leukemia cell (U937 excluded) were found to express VEGF in the supernanant. After exposure of HL-60 cells to VEGF ASODN at a concentration of 0.5, 1 and 5 micromol/L for 24 hours, the cell viability gradually dropped down to lower levels (P < 0.05 vs controls). After treatment of HL-60 cells with VEGF ASODN at a concentration of 1, 5 and 20 micromol/L for 24 hours, the VEGF levels in supernatants of target cells decreased (P < 0.05 vs controls). The patients with acute leukemia represented the higher levels of serum bFGF and VEGF than controls. Most of leukemia cell lines expressed bFGF and VEGF at different levels. It is concluded that bFGF and VEGF both have effects on regulations of angiogenesis in acute leukemia, but VEGF plays a pivotal role. VEGF-specific ASODN may have a role in them VEGF expression downregulated. Different results may be obtained in the evaluation of VEGF levels in the serum of patients with acute leukemia if different calculation methods are used. The methods reported can measure leukemia associated VEGF more accurately.