All.trans retinoic acid (RA) is the first highly effective differentiation inducing agent for remission induction in patients with acute promyelo.

All.trans retinoic acid (RA) is the first highly effective differentiation inducing agent for remission induction in patients with acute promyelo.. cytic leukemia. However, remissions are short-lived because the treatment fails to induce complete differentiation and fails to eradicate the malignant clone. To eliminate rapidly the malignant clone, in analogy with aggressive chemotherapy, the combination of potent differentiationand apoptosis inducing drugs working through different receptors and signal pathways may be useful. The active form of vitamin D3 (1,25-dihydroxyvitamin D3; 1,25(OH)2D3)inhibits proliferation and induces differentiation of myeloid leukemic cells. The 9.cis-RA, unlike all.trans-RA which binds only retinoic acid receptors, is a high affinity ligand for both retinoic acid receptors and retinoid X receptors. The aim of this study was to evaluate the therapeutic potential of combining a vitamin D3 analogue, 20.epi.22-oxa.24a,26a,27a. tri.homo-la,25(OH)2D3 (KH 1060), which belongs to the family of potent 20.epi.1,25(OH)2D3 analogues, with 9.cis.RA by assessing their effects on the proliferation, differentiation, and apoptosis of the human leukemia cell line HL-60 in vitro.Our data show that KB 1060 alone is a very potent inhibitor of clonal proliferation of IIL-60, but this effect is reversible, and that 9.cis.RA alone is a weak inhibitor of clonal proliferation of HL.60 cells. In contrast, the combination of 1(11 1060 and 9.cis.RA synergistically and irreversibly inhibited the clonal proliferation of HL.60 cells and induced apoptosis, as detected by morphological changes and DNA frag mentation. This combination also affected the expression of apoptosis related genes. The bcl.2 protein became nearly undetectable, and expres. sion of bax protein increased slightly (the bax:bcl-2 ratio was 14-fold higher than in untreated cells). Differentiation of treated HL-60 ceHs was assessed by their ability to produce superoxide, as measured by reduction of nitro blue tetrazolium, positive staining for a-naphthyl acetate esterase, phagocytosis, morphology, and analysis of membrane-bound differentia tion markers with two-color immunofluorescence. Treatment with the combination of KR 1060 and 9-cis.RA was a potent inducer of differen tiatlon of HL-60, with the cells developing a myelomonocytic phenotype. In summary, our data demonstrate that the combination ofboth KH 1060 and 9.cis.RA irreversibly and synergistically Inhibited clonal growth, induced 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. Received 1/3 1/96; accepted 6/5/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grants CA-42710, CA-43277, and CA70675-01 and the Depart ment of Defense; the Concern Foundation; CaP CURE Foundation; and the Parker Hughes Fund. Supported in part by a grant from the Deutsche Forschunsgemeinschaft (to E. E.). H. P. K. holds the Mark Goodson Endowed Chair of Oncology Research and is a member of the Johnson Cancer Center. 2 To whom requests for reprints should be addressed, at Cedars-Sinai Medical Center! UCLA School of Medicine, Davis Building 5033, 8700 Beverly Boulevard, Los Angeles, CA 90048. Phone: (310) 855-4609; Fax: (310) 392-3809. 3570 Combination of a Potent 20.-epi-Vitamin D3 Analogue (KH 1060) with 9-cis-Retinoic Acid Irreversibly Inhibits Clonal Growth, Decreases bcl-2 Expression, and Induces Apoptosis in HL-60 Leukemic Cells1 Elena Elstner,2 Mariana Linker-Israeli, Tehila Umiel, Jennifer Le, Isabelle Grilhier, Jonathan Said, I. Peter Shintaku, Stanislaw Krajewski, John C. Reed, Lise Binderup, and H. Phiffip Koeffler Division of Hematology.Oncology [E. E., T. U., J. L., 1. G., H. P. K.], Rheumatology IM. L-I.J, Departments of Medicine and Pathology [J. S., P. S.], UCLA, School of Medicine, Cedars.Sinai Medical Center, Los Angeles, California 90048; La Jolla Cancer Research Foundation, La Jolla, California 92037 [S. K., J. C. R.]; and Department of Biology. Leo Pharmaceutical Products, Ballerup. Denmark IL. B.]