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KMID : 0857020050200010016
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Kosin Medical Journal
2005 Volume.20 No. 1 p.16 ~ p.29
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Down-regulation of a Serine Proteinase Myeloblastin mRNA in HL-60 cells
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Lee Dae-Heui
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Abstract
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Background: Recent clinical studies have shown that a high proportion of patients with acute promyelocytic leukemia(APL) achieves complete remission after treatment with all-trans retinoic acid (ATRA). However, most patients who receive continuous treatment with ATRA relapse and develop ATRA-resistant leukmia. In this study, the author investigated the strategies to overcome ATRA resistance of acute promyelocytic leukemia (APL) cells by inducing the differentiation of dendritic cells (DCs) from human leukemic cell lines for the developtment of adoptive immunotherapy. Myeloblastin (mbn) was used as one of the indicators of differentiation in this study.
Methods: To study the biochemical and enzymatic charicteristics of the human myeloblastin the enzyme was extracted from human leukocytes and purified by a combination of Ultrogel AcA 54 and Bio-Rex 70 chromatographies. To evaluate the mbn protein expression in cells, anti-mbn antibody was prepared by two-step procedure including ammonium sulfate precipitation and DEAE-cellulose ion exchange chromatography. HL-60 cell differentiation was induced by the addition of all-trans retinoic acid (ATRA), dimethyl sulfoxide (DMSO), phorbol 12-myristate 13-acetate (PMA), cholecalcitriol (VD) to the media for 6 days and the expression of mbn mRNA and mbn protein were determined by RT-PCR method and ELISA, respectively.
HL-60 cells, K-562 cells, NC-37 and RPMI 7666 cells were cultured in RMPI 1640 supplemented with 10% fetal calf serum for 7 days, with various agents or ligands such as calcium ionophore (CI), Flt3-ligand (FL) and PMA to generate dendritic cells from the cell lines. A portion of ezch cell lines was havested and the rest of them was cultured in the new RPMI 1640 supplemented with 10% fetal calf serum, with Flt3-ligand for 7 days more. RNA was extracted and gene expression from each cell lines was determined by RT-PCR method. The morphology of the cells was evaluated from cytospin slide preparations with Wright’s stain.
Result: 3.8 mg of proteinase-3 was isolated from 67 ㎎ of leukocyte extract. 6g of anti-mbn polyclonal antibody was raised. PMA induced a significant inhibition of mbn mRNA expression in HL-60 cells. The cells exposed to ATRA or DMSO or VD show a little change in the mbn mRNA expression. Thus the terminal differention of HL-60 cells and K-562 cells by ATRA, DMSO, VD, and hemin was incomplete and a large fraction of the cells was in undifferentiated or premature states. The promyelocytic leukemic cell line HL-60, B lymphoblast cell lines RPMI 7666 and NC-37 could be induced to dendritic cells in vitro. Treatment of HL-60 with PMA resulted in the expression of myeloid-related DC phenotypes, while treatment of RMPI 7666 with FL and treatment of NC-37 with PMA and FL lead to the expression of lymphoid-related DC phenotypes.
Conclusion: In conclusion, myeloid-related DC phenotypes and lymphoid-related DC phenotypes can be generated from HL-60, NC-37 RPMI 7666 cell lines, respectively. These DC phenotypes can potentially be used as a cellular leukemia vaccine in vivo or to generate antileukemic T cells in vitro for adoptive immunotherapy.
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KEYWORD
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ATRA-resistant leukemia, Adoptive immunotherapy, Dendritic cells(DCs), HL-60 cells, phorbol 12-myristate
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