Induction of Apoptosis in Human Leukaemia Cells is Differentially Regulated by Vitamin D Derivatives and Retinoids

Abstract

The ability of 1,25-dihydroxyvitamin D₃ and two of its analogues, EB1089 and KH1060 to induce apoptosis in two leukaemia cell lines, HL-60 and U937 were examined. In addition, the effects of the retinoid 9-cis retinoic acid (9-cis RA) were assessed alone or in combination with the vitamin D compounds. In both the HL-60 and U937 cell lines none of the vitamin D derivatives alone were capable of inducing apoptosis, as assessed by flow cytometry and cell death ELISA techniques. In contrast, 9-cis RA induced apoptosis in HL-60 but not U937 cells. Co-treatment of HL-60 cells with the vitamin D compounds and 9-cis RA resulted in an enhanced induction of apoptosis, which was not observed with U937 cell co-treatment. Western analysis of bcl-2 and bax protein levels revealed that combinations of the vitamin D and retinoid compounds decreased bcl-2 in HL-60 but not U937 cells, suggesting a change in the bcl-2/bax heterodimeric ratio in the former which would favour apoptosis. Further Western analyses were performed to examine the regulation of vitamin D receptor (VDR), retinoid X receptor (RXR) and retinoic acid receptor (RAR) following single or co-treatment with their respective ligands. Studies revealed that 9-cis RA alone down-regulated RXR, and diminished VDR and RAR protein levels in the presence of the vitamin D compounds in both cell lines. However, in HL-60 cells the vitamin D derivatives were capable of preventing the further down-regulation of RXR in the presence of the retinoid. These results demonstrate cell specific responses to the induction of apoptosis and regulation of apoptosis related gene products by vitamin D and retinoid compounds, highlighting complex signalling interactions between their receptors.

Keywords:

• Vitamin D analogues
• apoptosis
• 9-cis retinoic acid
• VDR
• RXR
• bcl-2