[In vitro activation of bone marrow natural killer T cells of aplastic anemia patients].

OBJECTIVE: To investigate the quantitative and qualitative changes of TCRVα24(+)Vß11(+) natural killer T (NKT) cells from bone marrow (BM) of aplastic anemia (AA) after in vitro stimulation of α-galactosylceramide (α-Galcer). METHODS: NKT cells in the bone marrow mononuclear cells (BMMNCs) from either AA patients or healthy controls were enumerated with flow cytometry. BMMNCs were cultured in RPMI1640 medium supplemented with either α-Galcer and rhIL-2 or α-Galcer, rhIL-2 and rhG-CSF. The proliferative capacity of NKT cells was determined by NKT cell numbers before and after in vitro culture. Expression of intracellular IFNγ and IL-4 in activated NKT cells was analyzed with flow cytometry. RESULTS: In AA group, the percentage of NKT cells in BMMNCs was (0.19 ± 0.09)%. Addition of rhG-CSF into the α-Galcer/rhIL-2 culture medium resulted in significantly reduced expansion of NKT cells (67.45 ± 29.42-fold vs 79.91 ± 40.56 fold, P < 0.05). Meanwhile, addition of rhG-CSF reduced IFNγ positive NKT cells \[(37.45 ± 7.89)% vs (62.31 ± 14.67)%, P < 0.01\] and increased IL-4 positive NKT cells \[(55.11 ± 12.13)% vs (27.03 ± 9.88)%, P < 0.01\]. In healthy control group, the percentage of NKT cells in BMMNCs was (0.25 ± 0.12)%. Addition of rhG-CSF into the α-Galcer/rhIL-2 culture medium also significantly reduced expansion of NKT cells (97.91 ± 53.22-fold vs 119.58 ± 60.49-fold, P < 0.05), reduced IFNγ positive NKT cells \[(28.65 ± 10.63)% vs (50.87 ± 12.66)%, P < 0.01\], and increased IL-4 positive NKT cells \[(66.53 ± 14.96)% vs (31.11 ± 10.07)%, P < 0.01\]. CONCLUSION: Compared to those from healthy controls, BMMNCs from AA patiants have a reduced fraction of NKT cells, which possesses a decreased potential to expand in vitro in response to α-Galcer stimulation, and produce more IFNγ(+) NKT1 cells. rhG-CSF, in combination with α-Galcer, confers polarization of NKT cells towards IL-4(+) NKT2 subpopulation.

[Changes of gene expression profile in homoharringtonine-induced leukemia multi-drug resistant cell line K562/HHT].

OBJECTIVE: To study the resistant related molecules of human leukemia drug resistant K562 cells (K562/HHT) induced by homoharringtonine (HHT). METHODS: Gene expression profiles on K562/HHT, K562 and K562/HHT/RU486 (K562/HHT reversed by RU486) cells were detected by DNA microarray. The bone marrow tyrosine kinase gene in chromosome X (BMX) which changed dynamically among the three cells was confirmed by RT-PCR and Western blot. Then, BMX was transfected into K562 and K562/HHT cells, and the changes of daunorubicin (DNR) concentrations in these two cells were observed for BMX overexpression. RESULTS: As compared with K562, there were changes in 117 gene expressions in K562/HHT, 57 of which were up-regulated and 60 down-regulated. The mdrl gene was significantly up-regulated. When compared with K562/HHT, 50 significantly differently expressed genes were screened out in the K562/HHT/RU486 cells, of which up- and down-regulated genes were 13 and 37 respectively. These genes involved in drug resistance, cell signaling, cell differentiation, cell proliferation, transcription regulator, ion transport and so on. Four genes [NM-001721 (BMX), NM-031459 (SESN2), NM-033642 (FGF13) and AL-049309 (SFRS12)] expressed significantly differently in the two group cells, BMX gene expression was higher in K562/HHT, than in K562, but lower than in K562/HHT/RU486 as confirmed by RT-PCR and Western blot. After the plasmid pCI-neo-BMX was transfected into K562 and K562/HHT cells, DNR concentration was significantly lower (79.28 +/- 4.04, 29.84 +/- 2.67) than those before transfection (158.52 +/- 8.08, 58.58 +/- 6.53). CONCLUSION: BMX is associated with multi-drug resistance of K562/HHT cell line.

[Study of mifepristone on reversing multidrug resistance of leukemic cell line K562/A02].

OBJECTIVE: To study whether progestogen antagonist mifepristone could reverse multidrug resistance of K562/A02 cells and its mechanisms. METHODS: MTT was used to study the proliferation of K562/A02 cells and sensitivity of K562/A02 cells to ADM after 72 hours treatment with mifepristone. Flow cytometry was used to assay the expression of P-glycoprotein and the mean fluorescent intensity of intracellular daunorubicin. The expressions of apoptosis related proteins (bcl-2, Bax and caspase-3) were assayed by immunohistochemistry and the glucosylceramide synthase mRNA expression by RT-PCR before and after mifepristone treatment. RESULTS: MTT assay revealed that 2.5, 5.0 and 10 micromol/L mifepristone did not affect the proliferation of K562/A02 cells, but enhanced the sensitivity of K562/A02 cells to ADM, by 1. 68-, 4.17- and 10.71- fold increase, respectively. Expression of P-gp in K562/A02 cells was (49.03 +/- 5.32)%, and was decreased to (28.60 +/- 2.13)% (P < 0.01) after 10 micromol/L mifepristone treatment for 72 hours. and intracellular DNR accumulation in K562/A02 was (61.07 +/- 8.61)%, and was increased to (92.72 +/- 3.48)% (P < 0.01). After 10 micromol/L mifepristone treatment, the expression of bcl-2 protein was decreased from (56 +/- 9)% to (37 +/- 6)% (P < 0.05), Bax and caspase-3 proteins was increased from (40 +/- 5)% to (87 +/- 10)% (P < 0.01), and from (36 +/- 7)% to (89 +/- 6)% (P < 0.01) respectively. RT-PCR analysis revealed that expression of glucosylceramide synthase mRNA was higher in K562/A02 than in K562 cells, whereas 10 micromol/L mifepristone significantly down-regulated its expression in K562/A02 cells. CONCLUSION: Mifepristone at 10 micromol/L could dose-dependently reverse the multidrug resistance of K562/A02 cells. The possible mechanisms are related with decreasing the expression of P-gp, regulating the expression of apoptosis related proteins and decreasing the expression of glucosylceramide synthase.

[Improvement effect of bacterium derived oligonucleotides on maturation of K562/A02 cells derived dendritic cells].

OBJECTIVE: To study the maturation effect of CpG2006 and phosphodiester oligonucleotides on leukemia-derived dendritic cells. METHODS: Leukemia cells K562/A02 were induced into dendritic cells by rhGM-CSF and rhIL-4. After 7 days induction, the cell-morphology was observed, the immunophenotype of cells was detected by flow cytometry and the cell function was evaluated by allogeneic mixed lymphocyte reactions, CTL responses and secretion of IL-12 and IL-6. Then a CpG oligonucleotide CpG2006, two synthetic bacterial phosphodiester oligonucleotides A-ODN and T-ODN were added to these leukemia-derived DCs. Three days later, the DCs were re-detected by the above-mentioned methods. RESULTS: After induced by CpG2006, A-ODN or T-ODN, the leukemia-derived DCs with typical dendritic morphology were increased. The expressions of CD83, HLA-DR and CD86 were (65.5 +/- 8.4)%, (32.0 +/- 4.3)% and (18.6 +/- 3.2)% respectively in day 7 leukemia-derived DCs, raised to (88.9 +/- 3.6)%, (53.9 +/- 3.2)% and (39.9 +/- 7.3)% respectively after exposing CpG2006 for 3 days; increased to (97.0 +/- 5.3)%, (63.9 +/- 7.3)% and (40.2 +/- 7.4)% respectively after treated by A-ODN; and further increased to (93.26 +/- 4.65)%, (58.3 +/- 5.6)% and (36.2 +/- 6.8)% respectively after treated by T-ODN. These results was markedly different than unaffected cells did. These DCs induced by the above-mentioned three oligonucleotides could upregulate significantly the capacity for stimulating allogeneic T cells. They could also induce CTL to generate specific cytotoxic activity against K562/A02 cells. The secretion of IL-6 and IL-12 was increased remarkably. CONCLUSION: CpG2006, as well as two phosphodiester oligonucleotides can induce leukemia-derived DCs maturation.

Proteomic analysis on multi-drug resistant cells HL-60/DOX of acute myeloblastic leukemia.

Multi-drug resistance (MDR) is an important factor that causes treatment failure in acute leukemia. However, the full development mechanisms of MDR still await [corrected] investigation. The purpose of this study is to investigate differentially expressed proteins in the multi-drug resistant acute myeloblastic leukemia (AML) cell line HL-60/DOX and the drug sensitive cell line HL-60, and to identify new potential multi-drug resistant related molecules with the proteomic approach. Two-dimensional gel electrophoresis (2-DE) maps of the proteins, extracted from two AML cell lines, HL-60/DOX and HL-60, were established respectively. The extracted proteins were digested by enzymes and identified with the matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The data of the peptide mass fingerprinting (PMF) was matched with databases of proteomics available on the Internet. Results showed that 16 proteins were identified to be differentially expressed between HL-60/DOX and HL-60 cells. They involved the protein disulfide isomerase precursor (PDI), the proteasomes alpha1 and other proteins which are related to drug resistance or cell metabolism, but their functional significances are required further investigation. Nevertheless, it is clear that this proteomic approach for studing the biology and development of MDR is a prerequisite in leukemia.