Monocytic differentiation induced by side-chain modified analogs of vitamin D in ex vivo cells from patients with acute myeloid leukemia.

The differentiation-inducing potential of side-chain modified analogs of vitamins D, compared to the reference compound, 1,25-dihydroxyvitamin D3, was studied in blast cells from patients with acute myeloid leukemia and in cell lines. Analogs PRI-1906 and PRI-1907 showed increased cell-differentiation activities, PRI-1907 even at a very low concentration. Our study revealed a high variability of individual patients' blasts in their susceptibility to vitamin D analogs. The blasts of the patients with normal karyotype and with mutated NPM1 reacted to analogs with stronger differentiation than the blasts of the remaining patients, while the blasts with mutated FLT3 receptor reacted with weaker differentiation than the remaining blasts.

Regulation of Leukemic Cell Differentiation through the Vitamin D Receptor at the Levels of Intracellular Signal Transduction, Gene Transcription, and Protein Trafficking and Stability.

1α,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D) exerts its biological activities through vitamin D receptor (VDR), which is a member of the superfamily of steroid receptors, that act as ligand-dependent transcription factors. Ligated VDR in complex with retinoid X receptor (RXR) binds to regulatory regions of 1,25(OH)(2)D-target genes. 1,25(OH)(2)D is able to induce differentiation of leukemic blasts towards macrophage-like cells. Many different acute myeloid leukemia (AML) cell lines respond to 1,25(OH)(2)D by increasing CD14 cell surface receptor, some additionally upregulate CD11b and CD11c integrins. In untreated AML cells VDR protein is present in cytosol at a very low level, even though its mRNA is continuously expressed. Ligation of VDR causes protein stabilization and translocation to the cell nuclei, where it regulates transcription of target genes. Several important groups of genes are regulated by 1,25(OH)(2)D in HL60 cells. These genes include differentiation-related genes involved in macrophage function, as well as a gene regulating degradation of 1,25(OH)(2)D, namely CYP24A1. We summarize here the data which demonstrate that though some cellular responses to 1,25(OH)(2)D in AML cells are transcription-dependent, there are many others which depend on intracellular signal transduction, protein trafficking and stabilization. The final effect of 1,25(OH)(2)D action in leukemic cells requires all these acting together.

Studies on the mechanisms of superagonistic pro-differentiating activities of side-chain modified analogs of vitamin D2.

1,25-Dihydroxyvitamin D3 (1,25D) is implicated in many cellular functions including cell proliferation and differentiation, thus, exerting potential antitumor effects. A major limitation for therapeutic use of 1,25D are its potent calcemic and phosphatemic activities. Therefore, synthetic analogs of 1,25D for use in anticancer therapy should retain cell differentiating potential, with calcemic activity being reduced. Previously, we described pro-differentiating effects of vitamin D2 analogs with extended and branched side-chains. Analogs with side-chains extended by a pair of one (PRI-1906) or two carbon units (PRI-1907) displayed elevated cell-differentiating activity towards some acute leukemia cell lines (AML) in comparison to 1,25D. In this study, the potential mechanism of this superagonistic activity of the analogs was addressed. At first, possible differences in the expression of CYP24A1, a major catabolizing enzyme for vitamin D compounds and resulting differences in the degradation of analogs were investigated. In addition, interactions of the analogs with vitamin D receptor (VDR) and resulting activation of CCAAT-enhancer-binding protein ß (C/EBPß) were studied. The results obtained show that superagonistic pro-differentiating activities of analogs PRI-1906 and PRI-1907 do not seem to be caused by their altered catabolism, but most probably by altered interactions with VDR and resulting downstream proteins.

Opposite regulation of vitamin D receptor by ATRA in AML cells susceptible and resistant to vitamin D-induced differentiation.

Some leukemic cell lines can be driven to differentiate to monocyte-like cells by 1,25-dihydroxyvitamin D(3) (1,25D) and to granulocyte-like cells by all-trans retinoic acid (ATRA). Acute myloid leukemias (AMLs) are heterogeneous blood malignancies characterized by a block at various stages of hematopoietic differentiation and there are more than 200 known chromosome translocations and mutations in leukemic cells of patients diagnosed with AML. Because of the multiplicity in the genetic lesions causing the disease, AMLs are particularly difficult to treat successfully. In particular, various AML cells to a variable degree respond to 1,25D-based differentiation and only one type of AML undergoes successfully ATRA-based differentiation therapy. In this paper we describe that AML cell line KG-1 is resistant to 1,25D-induced monocytic differentiation, while sensitive to ATRA-induced granulocytic differentiation. We show that KG-1 cells have very low level of VDR protein and that expression of VDR mRNA is upregulated by ATRA. We show for the first time that this regulation is cell context-specific, because in another AML cell line, HL60, VDR mRNA is downregulated by ATRA. ATRA-induced VDR protein in cytosol of KG-1 cells can be further activated by 1,25D to induce monocytic differentiation of these cells.

Different susceptibilities to 1,25-dihydroxyvitamin D3-induced differentiation of AML cells carrying various mutations.

This study was designed to compare the differentiation-inducing potential of 1,25-dihydroxyvitamin D(3) (1,25D) with some analogs (VDAs) in a panel of acute myeloid leukemia (AML) cell lines and in blast cells isolated from patients with AML. Of the cell lines studied, HL60 proved to be the most sensitive to each of the differentiation-inducing agents when compared to THP-1, NB-4 and U-937 cell lines. Three of the VDAs tested (PRI-1906, PRI-2191 and PRI-2201) were similarly effective as 1,25D in all the cell lines tested. However, blast cells from AML showed a varying sensitivity towards 1,25D. For example, blast cells isolated from patients in which the whole or part of chromosome 7 was deleted were extremely sensitive to 1,25D and its analogs. In contrast, 1,25D failed to increase the expression of differentiation markers in blast cells isolated from patients carrying activating mutations in Flt3 gene. Since, the expression of vitamin D receptor (VDR) in cells with Flt3 mutations was increased to the same extent as in other AML cells this suggests that failure of these cells to differentiate lies downstream of the receptor. That blast cells with different cytogenetic abnormalities have dissimilar responses to 1,25D and its analogs, may have implications in the use of 1,25D as a 'differentiation therapy' for myeloid leukemias. The analog PRI-2191 (tacalcitol) was found to be the most potent in inducing patient's cells differentiation.