[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.


CONCLUSIONS
MG132 induces the expression of costimulatory molecule CD86 in the HL-60 cells, thus improving the proliferation of PBMNC.
Zhou
Yong-Ming
YM
Center for Stem Cell Amplification and Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Guo
Wei
W
Xue
Ke-Ying
KY
Zhou
Hao
H
Cheng
Yan-Xiang
YX
Guo
Tian-Nan
TN
Li
Hui-Yu
HY
Huang
Shi-Ang
SA
D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D018122:B7-1 Antigen / Q000235:genetics* / Q000378:metabolism; D051940:B7-2 Antigen / Q000235:genetics* / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D007938:Leukemia / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects / Q000528:radiation effects; D007976:Leupeptins / Q000494:pharmacology*; D061988:Proteasome Inhibitors; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018122:B7-1 Antigen; D051940:B7-2 Antigen; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D061988:Proteasome Inhibitors; D012333:RNA, Messenger; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde