COMBINATION OF A POTENT 20-EPI-VITAMIN-D-3 ANALOG (KH-1060) WITH 9-CIS-RETINOIC ACID IRREVERSIBLY INHIBITS CLONAL GROWTH, DECREASES BCL-2 EXPRESSION, AND INDUCES APOPTOSIS IN HL-60 LEUKEMIC-CELLS

All-trans retinoic acid (RA) is the first highly effective differentia tion-inducing agent for remission induction in patients with acute pro myelocytic leukemia, However, remissions are short-lived because the t reatment fails to induce complete differentiation and fails to eradica te the malignant clone. To eliminate rapidly the malignant clone, in a nalogy with aggressive chemotherapy, the combination of potent differe ntiation- and apoptosis-inducing drugs working through different recep tors and signal pathways may be useful, The active form of vitamin D-3 (1,25-dihydroxyvitamin D-3; 1,25(OH)(2)D-3) inhibits proliferation an d induces differentiation of myeloid leukemic cells. The 9-cis-RA, unl ike all-trans-RA which binds only retinoic acid receptors, is a high a ffinity ligand for both retinoic acid receptors and retinoid X recepto rs. The aim of this study was to evaluate the therapeutic potential of combining a vitamin D-3 analogue, 20-epi-22-oxa-24 alpha,26 alpha,27 alpha-tri-homo-1 alpha,25(OH)(2)D-3 (KH 1060), which belongs to the fa mily of potent 20-epi-1,25(OH)(2)D-3 analogues, with 9-cis-RA by asses sing their effects on the proliferation, differentiation, and apoptosi s of the human leukemia cell line HL-60 in vitro, Our data show that K H 1060 alone is a very potent inhibitor of clonal proliferation of HL- 60, but this effect is reversible, and that 9-cis-RA alone is a weak i nhibitor of clonal proliferation of HL-60 cells. In contrast, the comb ination of KH 1060 and 9-cis-RA synergistically and irreversibly inhib ited the clonal proliferation of HL-60 cells and induced apoptosis, as detected by morphological changes and DNA fragmentation, This combina tion also affected the expression of apoptosis-related genes, The bcl- 2 protein became nearly undetectable, and expression of bax protein in creased slightly (the bax:bcl-2 ratio was 14-fold higher than in untre ated cells), Differentiation of treated HL-60 cells was assessed by th eir ability to produce superoxide, as measured by reduction of nitro b lue tetrazolium, positive staining for alpha-naphthyl acetate esterase , phagocytosis, morphology, and analysis of membrane-bound differentia tion markers with two-color immunofluorescence. Treatment with the com bination of KH 1060 and 9-cis-RA was a potent inducer of differentiati on of HL-60, with the cells developing a myelomonocytic phenotype. In summary, our data demonstrate that the combination of both KH 1060 and 9-cis-RA irreversibly and synergistically inhibited clonal growth, in duced differentiation and apoptosis of HL-60 cells concomitantly with a very marked decreased expression of bcl-2, and increased the bax:bcl -2 ratio, This drug combination may have important therapeutic signifi cance.