[Role of MG132, a proteasome inhibitor, in inducing expression of costimulatory molecules CD86 in leukemic cells and its effect on allogeneic mixed lymphocyte reaction].

OBJECTIVE: To investigate the role of proteasome inhibitors MG132 in the inducing the expression of the costimulatory molecules CD80 and CD86 in leukemia cells and its effect on allogeneic mixed lymphocyte reaction. METHODS: Acute myelocytic leukemia cells of the line HL-60 and chronic myelocytic leukemia cells of the line K562 were cultured. 7-AAD staining and flow cytometry (FC) were used to examine the viability of the cells. MG132, a proteasome inhibitor, of the concentrations of 2 or 3 micromol/L was added into the culture fluid of HL-60 cells for 24 h and 48 h respectively and then annexin V/7-AAD staining and FC were used to detect the apoptosis of the cells. HL-60 and K562 cells treated with 1 micromol/L MG132 for 24 h and 48 h respectively, anti-CD80 and anti-CD86 antibodies were added, then FC was used to detect the expression of CD80 and CD86. The mRNA expression of CD86 in the HL-60 cells treated with 1 micromol/L MG132 was examined by RT-PCR. HL-60 and K562 cells were treated by 1 micromol/L MG132 for 48 h and then underwent irradiation of 75 Gy Co-60 to kill the cells with their antigenicity preserved. Peripheral blood mononuclear cells (PBMNC) of healthy volunteers, as reactive cells, were isolated and inoculated into the Co-60 treated HL-60 and K532 cells of different concentrations, as stimulating cells, for 5 d, CCK-8, a new agent to detect the cell viability, was added for 4 h, and then the A value of absorbance was measured at the wave length of 450 nm of enzyme labeling instrument. Control groups were set up for all tests. RESULTS: The cell viability rates of the HL-60 cell treated with 1 micromol/L MG132 for 24 h and 48 h were 92.95% and 85.87% respectively. The apoptotic rats of the HL-60 cells treated with MG132 were increased dose- and time-dependently. Before MG132 treatment K562 cells did not express CD86, and the CD86 expression of the HL-60 cells was up-regulated time-dependently (all P < 0.01). The mRNA expression of CD86 in the HL-60 treated with MG132 was up-regulated time-dependently (P < 0.01). CKK8 test showed that the proliferation level of PBMNC gradually increased along with the concentration of HL-60 cells treated with MG132 and reached its peak when the concentration of the HL-60 cells was 1 x 10(5) (P < 0.01). No remarkable proliferation of PBMNC was seen in the K562 groups no matter if the HL-60 cells had been treated with MG132. CONCLUSION: MG132 induces the expression of costimulatory molecule CD86 in the HL-60 cells, thus improving the proliferation of PBMNC.

[Differential expression profiles of MicroRNA during the development of human cord blood CD34(+)CD38(-) cells to CD34(+)CD38(+) cells].

To establish a basis for deep investigation of the role of microRNA (miRNA) in the regulation of hematopoiesis, differential expression profiles of miRNA between human cord blood CD34(+)CD38(-) and CD34(+)CD38(+) cells were analyzed. Mononuclear cells from cord blood (CB) of healthy donors were separated by Ficoll-Hypaque density gradients. CD34(+)CD38(-) and CD34(+)CD38(+) cells were sorted by using FACS Vantage SE. Their mRNA were then extracted and hybridized to miRNA microarray chip. The resulting data were analyzed with GeneSpring and informatics technique. The results showed that eleven miRNAs were found to be downregulated and 73 miRNAs to be upregulated by at least two-fold in the CD34(+)CD38(+) cells of CB, compared with the CD34(+)CD38(-) cells, which maintained CD34(+)CD38(-) cells' self-renewal and multiple lineage potential, that were defined as "stemness" miRNAs. 12 of the 84 genes (14.29%) were common to 33 hematopoietic-expressed miRNAs expressed by CD34(+) cells from both peripheral blood and bone marrow in Georgantas's study, which included 10 upregulated miRNAs (hsa-miR-23b, -26b, -92, -107, -130a, -181a, -197, -213, -222, -223) and 2 downregulated ones (hsa-miR-16a, -155). Some "stemness" miRNAs undergo CD34 antigen-like expression pattern during development and commmitted differeniation of hematopoietic stem cell/progenitors. Hematopoiesis-associated miRNA clusters and putative target genes could be found with informatics technique. It is concluded that the hematopoietic "stemness" miRNAs play important roles in normal hematopoiesis: miRNA expression profiles of hematopoietic stem cell/progenitors --> their gene expression profiles --> their self-renewal and lineage-commmitted differeniation.