title: Expression of costimulatory molecule CD86 in HL-60 cells induced by MG132 and its effect on allogeneic mixed lymphocyte reaction.
abstract: This study was aimed to elucidate the expression of costimulatory molecule CD80 and CD86 in HL-60 cells induced by proteasome inhibitor MG132 and its effect on allogeneic mixed lymphocyte reaction. Acute myelocytic leukemia cell line HL-60 and chronic myelocytic leukemia cell line K562 were cultured. The viability of the cells was measured by flow cytometry. Proteasome inhibitor MG132 at the concentrations of 2 or 3 µmol/L was used to stimulate the HL-60 cell cultured for 24 h and 48 h respectively, and the Annexin V/7-AAD staining and flow cytomotry were used to detect the apoptosis of the HL-60 cells. HL-60 and K562 cells were treated with 1 µmol/L MG132 for 24 h and 48 h respectively, then CD80 and CD86 antibodies were added, finally the expression of CD80 and CD86 was analysed by flow cytomery. The mRNA expression of CD86 in the HL-60 cells treated with 1 µmol/L MG132 was detected by RT-PCR. HL-60 and K562 cells were treated by 1 µmol/L MG132 and then underwent irradiation of 75 Gy (60)Co to kill the cells with their antigenicity preserved. Peripheral blood mononuclear cells (PBMNCs) of healthy volunteers, as reactive cells, were isolated and inoculated into the (60)Co irradiated HL-60 cells of different concentrations, as stimulating cells, CCK-8 was added and then the A value of absorbance was measured at the wave length of 450 nm in an enzyme labeling instrument. The results showed that the cell viability of the HL-60 cells treated with 1 µmol/L MG132 for 24 h an d 48 h was 92.95% and 85.87% respectively. The apoptotic rates of the HL-60 cells treated with MG132 increased in dose-and time-dependent manner. High-concentration of MG132 directly killed HL-60 cells. Before MG132 treatment K562 cells did not express CD86, but the CD86 expression of the HL-60 cells was up-regulated time-dependently after MG132 treatment (P < 0.01). The mRNA expression of CD86 in the HL-60 treated with MG132 was up-regulated time-dependently (P < 0.01). CCK-8 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 1×10(5) (P < 0.01). No remarkable proliferation of PBMNC was observed in the K562 groups no matter if the HL-60 cells had been treated with MG132. It is concluded that the high concentration of MG132 can directly kill HL-60 cells, low-concentration of MG132 can induce the expression of costimulatory molecule CD86 in HL-60 cells, also can improve the proliferation of PBMNC.
authors.affiliation: Department of Hematology, The Affiliated Tianyou Hospital, Wuhan University of Science and Technology, Wuhan 430064, Hubei Province, China.
authors.forename: Mei-Xia
authors.initials: MX
authors.lastname: Yu
authors.affiliation: Division of Nephrology, Department of Internal Medicine, The Third Affiliated Hospital of Sun Yat-San Unversity, Guangzhou 510630, Guangdong Province, China.
authors.forename: Xun
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Hematology, The Affiliated Tianyou Hospital, Wuhan University of Science and Technology, Wuhan 430064, Hubei Province, China. E-mail: zhym112@126.com.
authors.forename: Yong-Ming
authors.initials: YM
authors.lastname: Zhou
authors.affiliation: Department of Gynecology and Obstetrics, The Affiliated People's Hospital of Hubei Province, Wuhan University, Wuhan 430060, Hubei Province, China.
authors.forename: Yan-Xiang
authors.initials: YX
authors.lastname: Cheng
authors.affiliation: Central Laboratory, The Affiliated Tianyou Hospital, Wuhan University of Science and Technology, Wuhan 430064, Hubei Province, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Cheng
authors.affiliation: Department of Hematology, The Affiliated Tianyou Hospital, Wuhan University of Science and Technology, Wuhan 430064, Hubei Province, China.
authors.forename: Yu-Zhen
authors.initials: YZ
authors.lastname: Qiu
authors.affiliation: Department of Hematology, The Affiliated Tianyou Hospital, Wuhan University of Science and Technology, Wuhan 430064, Hubei Province, China.
authors.forename: Xiao-Lei
authors.initials: XL
authors.lastname: Xing
authors.affiliation: Department of Hematology, The Affiliated Tianyou Hospital, Wuhan University of Science and Technology, Wuhan 430064, Hubei Province, China.
authors.forename: Chun-Hong
authors.initials: CH
authors.lastname: Yao
authors.affiliation: Department of Hematology, The Affiliated Tianyou Hospital, Wuhan University of Science and Technology, Wuhan 430064, Hubei Province, China.
authors.forename: Ru-Jun
authors.initials: RJ
authors.lastname: Bai
mesh_terms: D017209:Apoptosis; D051940:B7-2 Antigen / Q000276:immunology*; D002470:Cell Survival; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D007963:Leukocytes, Mononuclear / Q000187:drug effects; D007976:Leupeptins / Q000494:pharmacology*; D007959:Lymphocyte Culture Test, Mixed; D061988:Proteasome Inhibitors / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation
chemical_list: D051940:B7-2 Antigen; C497964:CD86 protein, human; D007976:Leupeptins; D061988:Proteasome Inhibitors; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde