Blast maturity and CD34 expression determine the effects of the differentiating agents KH1060 and 9-cis-retinoic acid on the differentiation and clonogenicity of non-M3 acute myeloid leukaemia cells

Abstract

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 × 10 −7 M 9-cRA and 5 × 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.

Membrane and Intracellular Platelet‐Activating Factor Receptor Expression in Leukemic Blasts of Patients with Acute Myeloid and Lymphoid Leukemia

“…Contrary studies have reported that M2‐type AML blasts are efficiently responsive [21] or weakly responsive [22,, 23] to this molecule. Blast maturity and CD34 expression were reported to determine the effect of a vitamin D analogue [24]. In this study, vitamin D 3 (but also DMSO and M‐CSF) did not induce AML2 blast differentiation (data not shown).…”

Induction of apoptosis by vitamin D3 analogue EB1089 in NCI‐H929 myeloma cells via activation of caspase 3 and p38 MAP kinase

“…Similarly, our data showed that the induction of apoptosis was accompanied by the down‐regulation of Bcl‐2 protein, supporting the theory that alteration of Bcl‐2 is directly or indirectly involved in the apoptotic effect of EB1089 in NCI‐H929 cells. However, no detectable change in Bcl‐2 expression was noted in primary acute myeloid leukaemia cells during apoptosis by vitamin D 3 compounds ( Pallis et al , 1998 ). On the contrary, the down‐regulation of Bcl‐2 by vitamin D 3 compounds could not trigger apoptosis in MDA‐MB‐231 breast cancer cells ( Elstner et al , 1995 ).…”

“…Our preliminary results demonstrated that EB1089‐mediated G 1 phase arrest resulted from the induction of cyclin‐dependent kinase inhibitor p27 in association with reduction of CDK2 activity without detectable change of p53 and p21 proteins (data not shown). Vitamin D 3 compounds are known to induce apoptosis in a variety of malignant cells ( James et al , 1996 , 1998; Puthier et al , 1996 ; Danielsson et al , 1997 , 1999; Fife et al , 1997 ; Simboli‐Campbell et al , 1997 ; Wang & Studzinski, 1997; Baudet et al , 1998; Pallis et al , 1998 ). Our study also showed that apoptosis was markedly induced in NCI‐H929 cells exposed to 10 −7 m EB1089 for 72 h. To gain insight into the molecular mechanisms involved in apoptosis by EB1089, expression of the proapoptotic protein Bcl‐2 was assessed.…”

“…Therefore, investigators have focused on the synthesis of potent analogues of 1,25(OH) 2 D 3 . Vitamin D 3 analogues inhibit the clonal growth and induce the apoptosis of many types of malignant cells ( Colston et al , 1992 ; Naveilhan et al , 1994 ; Skowronski et al , 1995 ; Wali et al , 1995 ; Yu et al , 1995 ; James et al , 1996 , 1998; Lee et al , 1996 ; Puthier et al , 1996 ; Danielsson et al , 1997 ; Fife et al , 1997 ; Simboli‐Campbell et al , 1997 ; Wang & Studzinski, 1997; Pallis et al , 1998 ; Evans et al , 1999 ). However, the signalling transduction pathway or pathways that lead to apoptosis in response to vitamin D 3 in these cells was not well understood.…”

Catalytic asymmetric syntheses and biological activities of singly dehydroxylated 19-nor-1α,25-dihydroxyvitamin D3 A-ring analogs in cancer cell differentiation and apoptosis

“…Although the stereochemistry of hydroxyl groups at C-1 and C-3 in the A-ring provides the key structural motifs responsible for both biological activities, it is unclear how the 19-nor-1α,25(OH) 2 D 3 analogs mediate these unique biological responses. It has been reported that 1α,25(OH) 2 D 3 protects HL-60 cells from drug-induced apoptosis [40], and also reduces the apoptotic effects of retinoid N-(4-hydroxyphenyl)-all-trans-retinamide [47]. Because antisense inhibition of VDR expression induces apoptosis in U937 cells [48], it has been suggested that the anti-apoptotic effect of 1α,25(OH) 2 D 3 is mediated via binding to VDR.…”