calcitriol and Leukemia--Myeloid--Acute

(1) Hematological malignancies are characterized by an immortalization, uncontrolled proliferation of blood cells and their differentiation block, followed by the loss of function. The primary goal in the treatment of leukemias is the elimination of rapidly proliferating leukemic cells (named blasts). However, chemotherapy, which removes proliferating blasts, also prevents the remaining immune cells from being activated. Acute leukemias affect elderly people, who are often not fit to survive aggressive chemotherapy. Therefore, there is a need of milder treatment, named differentiation therapy, which might simulate the immune system of the patient. 1,25-Dihydroxyvitamin D, or low-calcemic analogs of this compound, were proposed as supporting therapy in acute leukemias. (2) Bone marrow blasts from patients with hematological malignancies, and leukocytes from healthy volunteers were ex vivo exposed to 1,25-dihydroxyvitamin D, and then their genomes and transcriptomes were investigated. (3) Our analysis indicates that 1,25-dihydroxyvitamin D regulates in blood cells predominantly genes involved in immune response, such as

Topics: Aged; Blood Cells; Cell Differentiation; Dihydroxycholecalciferols; Hematologic Neoplasms; Humans; Leukemia, Myeloid, Acute; Leukocytes

Many malignancies are driven by mutations within the gene for fibroblast growth factor receptor 1 (FGFR1). Previously, we have shown that signal transduction from the FOP2-FGFR1 fusion protein in acute myeloid leukemia KG1 cells is responsible for a low level of expression of the vitamin D receptor gene. In this paper, we address whether other fibroblast growth factor receptors regulate the vitamin D receptor (VDR) gene. We used the human myeloid leukemia U937 and HL60 cells, the bone cancer cell line U2OS, and cell transfection methods to answer the question. For myeloid leukemia cells, overexpression of FGFRs 1-3 genes caused a shift towards monocytic differentiation; this was extracellular regulated kinase (Erk) 1,2-dependent. Overexpression of FGFRs 1-3 genes also upregulated expression of the VDR gene, further sensitizing these cells to 1,25-dihydroxyvitamin D-induced monocyte differentiation. When we increased expression in bone cells, fibroblast growth factor receptors did not upregulate VDR gene expression, nor influence the activity of VDR. Fibroblast growth factor receptors are overexpressed in many neoplasms. Therefore, it may be reasonable to use vitamin D analogs to treat these cancers, to activate VDR and drive cell differentiation.

Topics: Cell Differentiation; Dihydroxycholecalciferols; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Receptors, Calcitriol; Receptors, Fibroblast Growth Factor

Vitamin D receptor (VDR) is present in multiple blood cells, and the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is essential for the proper functioning of the immune system. The role of retinoic acid receptor α (RARα) in hematopoiesis is very important, as the fusion of RARα gene with PML gene initiates acute promyelocytic leukemia where differentiation of the myeloid lineage is blocked, followed by an uncontrolled proliferation of leukemic blasts. RARα takes part in regulation of

Topics: Animals; Blood Cells; Cell Differentiation; Cell Line, Tumor; Cells, Cultured; Gene Expression Regulation; Hematopoiesis; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Mice; Mice, Inbred C57BL; Receptors, Calcitriol; Retinoic Acid 4-Hydroxylase; Tretinoin; Vitamin D

Recent clinical trials aimed at improved treatment of AML by administration of vitamin D derivatives showed unremarkable results, suggesting development of vitamin D resistance in patients' AML blasts. Since mechanisms of vitamin D resistance are not clear, we studied 40AF cells, a subline of HL60 cells that can proliferate in the presence of 1α,25-dihydroxyvitamin D₃ (1,25D). We found that mRNA and protein levels of HPK1, an upstream MAP4 kinase, are dramatically increased in 40AF cells, and HPK1 protein is further increased when the 1,25D resistance of 40AF cells is partially reversed by the addition of carnosic acid and p38MAPK inhibitor SB202190 (DCS cocktail). Knockdown of HPK1 reduces 1,25D/DCS-induced differentiation of both 1,25D-sensitive HL60 and U937 cells and 1,25D-resistant 40AF cells, but the effect of HPK1 knockdown on differentiation-associated G 1 arrest is more apparent in the resistant than the sensitive cells. To explain why 40AF and the intrinsically vitamin D-resistant KG-1a cells can proliferate in the presence of vitamin D, we found that the cleaved HPK1 fragment (HPK1-C) level is high in 40AF and KG-1a cells, but when differentiation is induced by DCS, HPK1-C decreases while full-length (FL)-HPK1 increases. Accordingly, inhibition of proteolysis with the pan-caspase inhibitor Q-VD-OPh reduced HPK1 cleavage and enhanced DCS-induced differentiation of 40AF cells. The results indicate that FL-HPK1 is a positive regulator of vitamin D-induced differentiation in AML cells, but the cleaved HPK1 fragment inhibits differentiation. Thus, high HPK1 cleavage activity contributes to vitamin D resistance, and HPK1 has a dual role in AML cell differentiation.

Topics: Abietanes; Amino Acid Chloromethyl Ketones; Cell Cycle Checkpoints; Cell Differentiation; Cell Line, Tumor; Drug Resistance, Neoplasm; HL-60 Cells; Humans; Imidazoles; Leukemia, Myeloid, Acute; p38 Mitogen-Activated Protein Kinases; Plant Extracts; Protein Serine-Threonine Kinases; Pyridines; Quinolines; RNA Interference; RNA, Messenger; RNA, Small Interfering; U937 Cells; Vitamin D

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.

Topics: Cell Differentiation; Cytosol; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Receptors, Calcitriol; Steroid Hydroxylases; Tretinoin; Tumor Cells, Cultured; Vitamin D; Vitamin D3 24-Hydroxylase

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.

Topics: Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Separation; Chromosomes, Human, Pair 7; Flow Cytometry; HL-60 Cells; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Myeloid, Acute; Mutation; Receptors, Calcitriol; Vitamin D

Acute myelogenous leukemia (AML) is a disease characterized by dysregulated cell proliferation associated with impaired cell differentiation, and current treatment regimens rarely save the patient. Thus, new mechanism-based approaches are needed to improve prognosis of this disease. We have investigated in preclinical studies the potential anti-leukemia use of the plant-derived polyphenol Silibinin (SIL) in combination with 1,25-dihydroxyvitamin D3 (1,25D). Although most of the leukemic blasts ex vivo responded by differentiation to treatment with this combination, the reasons for the absence of SIL-1,25D synergy in some cases were unclear. Here we report that failure of SIL to enhance the action of 1,25D is likely due to the SIL-induced increase in the activity of differentiation-antagonizing cell components, such as ERK5. This kinase is under the control of Cot1/Tlp2, and inhibition of Cot1 activity by a specific pharmacological inhibitor 4-(3-chloro-4-fluorophenylamino)-6-(pyridin-3-yl-methylamino-3-cyano-[1-7]-naphthyridine, or by Cot1 siRNA, increases the differentiation by SIL/1,25D combinations. Conversely, over-expression of a Cot1 construct increases the cellular levels of P-ERK5, and SIL/1,25D-induced differentiation and cell cycle arrest are diminished. It appears that reduction in ERK5 activity by inhibition of Cot1 allows SIL to augment the expression of 1,25D-induced differentiation promoting factors and cell cycle regulators such as p27 (Kip1) , which leads to cell cycle arrest. This study shows that in some cell contexts SIL/1,25D can promote expression of both differentiation-promoting and differentiation-inhibiting genes, and that the latter can be neutralized by a highly specific pharmacological inhibitor, suggesting a potential for supplementing treatment of AML with this combination of agents.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p27; G1 Phase; Humans; Leukemia, Myeloid, Acute; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase 7; Proto-Oncogene Proteins; RNA Interference; RNA, Small Interfering; Silybin; Silymarin; Up-Regulation; Vitamin D