kh-1060 and Leukemia--Promyelocytic--Acute

The vitamin D analogue KH1060 and the retinoids all-trans retinoic acid (ATRA), 9-cis-retinoic acid (9-cRA) and 4-hydroxyphenyl retinamide (4-HPR) induce differentiation and/or apoptosis and inhibit clonal growth of acute promyelocytic leukaemia cells. We have studied the effects of these agents in vitro on cells from 12 patients with other forms of acute myeloblastic leukaemia (AML). Treatment with KH1060 (10(-6) M) caused decreases in cell viability in suspension culture to a median of 44% of control values (P=0.02). However, retinoids had little effect. Subsequent clonal growth in semi-solid medium was inhibited to 5% (median) of control with 10(-6) M KH 1060 (P=0.03) and to 73% with 10(-6) M 9-cRA (P=0.01). Further inhibition of clonal growth by the combination of 5 x 10(-7) M 9-cRA and 5 x 10(-7) M KH1060 was only noted in one case. Following the primary suspension culture, cells from 6/6 CD34 positive samples grew in semi-solid cultures without analogues, whereas cells from 3/6 CD34 negative cultures grew. 10(-6) M KH1060 completely abolished colony growth in all three CD34 negative samples and 10(-6) M 9-cRA inhibited the number of colonies to a median of 11% of control values. In the six CD34 positive samples median colony growth was inhibited to 36% of control values by KH1060 and to 83% of control values by 9-cRA. CD11b expression was increased by 210% (median) with 9-cRA and by 90% (median) with KH1060 in early to intermediate myeloblast (M0, M1, M2) clones. A different pattern was noted in more mature (M4, M5, M6) clones: here there was little or no increase in CD11b expression induced by retinoids or KH1060, but the ratio of apoptotic to viable CD11b+ cells, measured by CD11b/7-AAD double staining, was increased in 6/6 cases treated with KH1060 or the combination of 9-cRA and KH 1060, and in 5/6 cases treated with 9-cRA. No overall significant change in bcl-2 or bax expression on G0/G1 cells was found after 3 days' suspension culture with the analogues. However bcl-x was downregulated in G0/G1 cells treated with KH1060 (median bcl-x relative fluorescence intensity = 45.3 in cells treated with KH1060, compared with 65.7 in control wells, P=0.028). We conclude that CD34+ samples are relatively resistant to the growth inhibition induced by KH1060 and 9-cRA. However, downregulation of bcl-x in cells which have survived treatment with KH1060 may increase the susceptibility of the remaining leukaemic cells to cytotoxic drugs.

Topics: Alitretinoin; Antigens, CD34; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Calcitriol; Cell Differentiation; Cell Division; Flow Cytometry; Humans; Leukemia, Promyelocytic, Acute; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tretinoin; Tumor Cells, Cultured

Patients with acute promyelocytic leukemia (APL) usually relapse after all-trans retinoic acid (RA) treatment because this therapy fails to eradicate the malignant clone. Our data showed that KH 1060 and other 20-epi vitamin D3 analogs alone were potent inhibitors of clonal growth of NB4 cells, an APL cell line (ED50, approximately 5 x 10(-11) M). The combination of KH 1060 and 9-cis-RA synergistically and irreversibly enhanced this effect. Neither KH 1060 nor 9-cis-RA (10(-6) M, 3 d) were strong inducers of differentiation of NB4 cells. However, 98% of the cells underwent differentiation to a mature phenotype with features of both granulocytes and monocytes after exposure to a combination of both compounds. Apoptosis only increased after incubation of NB4 cells with 9-cis-RA alone (28%) or with a combination of 9-cis-RA plus KH1060 (32%). Immunohistochemistry showed that the bcl-2 protein decreased from nearly 100% of the wild-type NB4 cells to 2% after incubation with a combination of KH 1060 and 9-cis-RA, and the bax protein increased from 50% of wild-type NB4 cells to 92% after culture with both analogs (5 x 10(-7) M, 3 d). Western blot analysis paralleled these results. Studies of APL cells from one untreated individual paralleled our results with NB4 cells. Taken together, the data demonstrated that nearly all of the NB4 cells can be irreversibly induced to differentiate terminally when exposed to the combination of KH 1060 and 9-cis-RA.

Topics: Antigens, Differentiation; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Calcitriol; Cell Differentiation; Cell Division; Clone Cells; Dose-Response Relationship, Drug; Drug Synergism; Female; Humans; Leukemia, Promyelocytic, Acute; Middle Aged; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tretinoin; Tumor Cells, Cultured