Studies On The All Trans Retinoic Acid-Induced Differentiation Of Human Promyelocytic Leukemia Cell Line HL-60 And Its Mechanism Of Action

All trans retinoic acid(RA) is a potent inducer which induces terminal difTerentiation of HL- 60 promyelocytic leukemia cell line along granulocytic lineage and an attractive therapeutic agent for acute promyelocytic leukemia (APL). Therefore, the mechanism of action of RA is of special interest in the field of differentiation therapy. In this thesis, a retinoic acid -resistant HL-60 subclone (assigned HL-60/ RA) is characterized and used to explore the mechanism of action of RA by studying the differential response of HL-60 / RA and its parental HL-60 cells to RA.A retinoic acid-resistant subclone HL-60 / R A was selected by continuously culturing HL-60 cells in the medium contained increasing concentrations of RA(lnM-1μM) for 6 months. Then these cells were cloned by soft agar clonogenic assay. The EC50 for morphology differentiation(EC50M) was 6.8nM and EC50 for NBT reduction (EC50N) was 5.0nM for HL-60 cells; The EC50M was 26μM and EC50N was more than 10μM for RA-resistant HL-60 / RA cell line. These resistant characteristics remain unchanged although the HL-60/RA cells have been continuously cultured in retinoic acid-free medium for about 1 year.The experiment data demonstrated that HL-60 cells exhibited NBT reduction (a functional granulocytic differentiation marker), when they were exposed to 1-10μM of some new retinoid compounds (such as R8923) for 5 days or 2% DMSO for 5 days, while the HL-60/ RA cells showed markedly decreased functional change upon the same treatment. However, when HL-60/ RA cells were exposed to 2μM TPA for 3-5 days, they displayed a morphological and functional difTerentiation along monocyte-macrophage pathway. These results suggest that HL- 60/RA is resistant to inducers for granulocytic differentiation rather than inducers for monocyte-macrophage differentiation.By means of MTT assay, it was observed that the HL-60 and HL-60 / RA showed approximately equivalant IC50 for the cytotoxic effects of cytosine arabinoside, harringtonine and homoharring -tonine.About 50% HL-60/ RA cells exhibited NBT reduction when they were exposed to 1μM RA in combination with 1-10μM calcium antagonists such as nimodipinc, vcrapamil. It is advisable that these agents could be tested to overcome the differentiation resistance to RA on APL patients.We have compared the responses of HL-60 and HL-60/ RA to RA in the follwing...