19-NOR VITAMIN-D ANALOGS - A NEW CLASS OF POTENT INHIBITORS OF PROLIFERATION AND INDUCERS OF DIFFERENTIATION OF HUMAN MYELOID-LEUKEMIA CELL-LINES

We have studied the in vitro biological activities and mechanisms of a ction of 1,25-dihydroxyvitamin D-3 (1,25D(3)) and nine potent 1,25D(3) analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and K asumi-1), The common novel structural motiff for almost all the analog s included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentra tion of analogs producing a 50% clonal inhibition (ED50) ranged betwee n 1 x 10(-9) to 4 x 10(-11) mol/L when using the HL-60 cell line, The most active compound ,25(OH)(2)-16,23E-diene-26-trifluoro-19-calcifero l (Ro 25-9716)] had an ED50 Of 4 x 10-11 mol/L; in contrast, the 1,25D 3 produced an ED50 Of 10(-9) mol/L with the HL-60 target cells. Ro 25- 9716 (10(-9) mol/L, 3 days) was a strong inducer of myeloid differenti ation because it caused 92% of the HL-60 cells to express CD11b and 75 % of these cells to reduce nitroblue tetrazolium (NBI). This compound (10(-8) mol/L, 4 days) also caused HL-60 cells to arrest in the G(1) p hase of the cell cycle (88% cells in G(1) v 48% of the untreated contr ol cells), The p27(kip-1), a cyclin-dependent kinase inhibitor which i s important in blocking the cell cycle, was induced more quickly and p otently by Ro 25-9716 (10(-7) mol/L, 0 to 5 days) than by 1,25D3, sugg esting a possible mechanism by which these analogs inhibit proliferati on of leukemic growth. The NB4 promyelocytic leukemia cells cultured w ith the Ro 25-9716 were also inhibited in their clonal proliferation ( ED50, 5 x 10-11 mol/L) and their expression of CD11b was enhanced (80% positive [10-9 mol/L, 4 days] v 27% untreated NB4 cells). Moreover, t he combination of Ro 25-9716 (10(-9) mol/L) and all-trans retinoic aci d (ATRA, 10-7 mol/L) induced 92% of the NB4 cells to reduce NET, where as only 26% of the cells became NET positive after a similar exposure to the combination of 1,25D3 and ATRA, Surprisingly, Ro 25-9716 also i nhibited the clonal growth of poorly differentiated leukemia cell line s (RA-res HL-60 [ED50, 4 x 10(-9) mol/L] and Kasumi-1 [ED50, 5 x 10(-1 0) mol/L]). For HL-60 cells, Ro 25-9716 markedly decreased the percent of the cells in S phase of the cell cycle and increased the expressio n of the cyclin-dependent kinase inhibitor, p27(kip-1). In summary, 19 -nor vitamin D-3 compounds strongly induced differentiation and inhibi ted clonal proliferation of various myeloid leukemia cell lines, sugge sting a therapeutic niche for their use in myeloid leukemia. (C) 1998 by The American Society of Hematology.