cholecalciferol and Leukemia--Myeloid--Acute

The important role of cell cycle regulatory molecules in all trans-retinoic acid (ATRA)- and vitamin D3-induced growth inhibition and differentiation induction has been intensively studied in both acute myeloid leukaemia primary cells and a variety of leukaemia cell lines. Cyclin-dependent kinases (CDK)-activating kinase has been demonstrated to interact with retinoic acid receptor (RAR)α in acute promyelocytic leukaemia cells, and inhibition of CDK-activating kinase by ATRA causes hypophosphorylation of PML-RARα, leading to myeloid differentiation. In many cases, downregulation of CDK activity by ATRA and vitamin D3 is a result of elevated p21- and p27-bound CDKs. Activation of p21 is regulated at the transcriptional level, whereas elevated p27 results from both (indirectly) transcriptional activation and post-translational modifications. CDK inhibitors (CKIs) of the INK family, such as p15, p16 and p18, are mainly involved in inhibition of cell proliferation, whereas CIP/KIP members, such as p21, regulate both growth arrest and induction of differentiation. ATRA and vitamin D3 can also downregulate expression of G1 CDKs, especially CDK2 and CDK6. Inhibition of cyclin E expression has only been observed in ATRA- but not in vitamin D3-treated leukaemic cells. In vitro, not only dephosphorylation of pRb but also elevation of total pRb is required for ATRA and vitamin D3 to suppress growth and trigger their differentiation. Finally, sharp reduction in c-Myc has been observed in several leukaemia cell lines treated with ATRA, which may regulate expression of CDKs and CKIs.

Topics: Cell Differentiation; Cholecalciferol; Cyclin-Dependent Kinase Inhibitor Proteins; Cyclin-Dependent Kinases; Humans; Leukemia, Myeloid, Acute; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Tretinoin

Topics: Animals; Bone Resorption; Calcitriol; Cell Line; Cell Transformation, Neoplastic; Cholecalciferol; Culture Techniques; Gene Expression Regulation; Granulocytes; Humans; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Lymphoma; Mice; Monocytes; Oncogenes; Transcription, Genetic

Several studies have suggested that low 25(OH) vitamin D3 levels may be prognostic in some malignancies, but no studies have evaluated their impact on treatment outcome in patients with acute myeloid leukemia (AML).. Vitamin D levels were evaluated in 97 consecutive, newly diagnosed, intensively treated patients with AML. MicroRNA expression profiles and single nucleotide polymorphisms (SNPs) in the 25(OH) vitamin D3 pathway genes were evaluated and correlated with 25(OH) vitamin D3 levels and treatment outcome.. Thirty-four patients (35%) had normal 25(OH) vitamin D3 levels (32-100 ng/mL), 34 patients (35%) had insufficient levels (20-31.9 ng/mL), and 29 patients (30%) had deficient levels (<20 ng/mL). Insufficient/deficient 25(OH) vitamin D3 levels were associated with worse relapse-free survival (RFS) compared with normal vitamin D3 levels. In multivariate analyses, deficient 25(OH) vitamin D3 , smoking, European Leukemia Network genetic group, and white blood cell count retained their statistical significance for RFS. Several microRNAs and SNPs were associated with 25(OH) vitamin D3 levels, although none remained significant after multiple test corrections; one 25(OH) vitamin D3 receptor SNP, rs10783219, was associated with a lower complete remission rate (P = .0442) and with shorter RFS (P = .0058) and overall survival (P = .0011).. It remains to be determined what role microRNA and SNP profiles play in contributing to low 25(OH) vitamin D3 level and/or outcome and whether supplementation will improve outcomes for patients with AML.

Topics: Adult; Aged; Aged, 80 and over; Calcifediol; Cholecalciferol; Female; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid, Acute; Male; MicroRNAs; Middle Aged; Neoplasm Recurrence, Local; Polymorphism, Single Nucleotide; Prognosis; Receptors, Calcitriol; Transcriptome; Treatment Outcome

By means of an unbiased, automated fluorescence microscopy-based screen, we identified the epidermal growth factor receptor (EGFR) inhibitors erlotinib and gefitinib as potent enhancers of the differentiation of HL-60 acute myeloid leukemia (AML) cells exposed to suboptimal concentrations of vitamin A (all-trans retinoic acid, ATRA) or vitamin D (1α,25-hydroxycholecalciferol, VD). Erlotinib and gefitinib alone did not promote differentiation, yet stimulated the acquisition of morphological and biochemical maturation markers (including the expression of CD11b and CD14 as well as increased NADPH oxidase activity) when combined with either ATRA or VD. Moreover, the combination of erlotinib and ATRA or VD synergistically induced all the processes that are normally linked to terminal hematopoietic differentiation, namely, a delayed proliferation arrest in the G0/G1 phase of the cell cycle, cellular senescence, and apoptosis. Erlotinib potently inhibited the (auto)phosphorylation of mitogen-activated protein kinase 14 (MAPK14, best known as p38(MAPK)) and SRC family kinases (SFKs). If combined with the administration of ATRA or VD, the inhibition of p38(MAPK) or SFKs with specific pharmacological agents mimicked the pro-differentiation activity of erlotinib. These data were obtained with 2 distinct AML cell lines (HL-60 and MOLM-13 cells) and could be confirmed on primary leukemic blasts isolated from the circulation of AML patients. Altogether, these findings point to a new regimen for the treatment of AML, in which naturally occurring pro-differentiation agents (ATRA or VD) may be combined with EGFR inhibitors.

Topics: Antigens, CD34; CD11b Antigen; Cell Cycle Checkpoints; Cell Differentiation; Cholecalciferol; ErbB Receptors; Erlotinib Hydrochloride; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Mitogen-Activated Protein Kinase 14; Phosphorylation; Protein Kinase Inhibitors; Quinazolines; src-Family Kinases; Tretinoin; Tumor Cells, Cultured

The leukaemia-specific fusion oncoprotein RUNX1/RUNX1T1 (AML1/ETO), resulting from the chromosomal translocation (8;21) in acute myeloid leukaemia (AML), imposes a striking genotype-phenotype relationship upon this distinct subtype of AML, which is mediated by multiple, co-ordinate downstream effects induced by this chimeric transcription factor. We previously identified the LAT2 gene, encoding the adaptor molecule LAT2 (NTAL, LAB), which is phosphorylated by KIT and has a role in mast cell and B-cell activation, as a target of the repressor activity of RUNX1/RUNX1T1. These results were confirmed and extended by demonstrating downregulation of the LAT2 protein in response to conditional RUNX1/RUNX1T1 expression, and its absence in primary AML with the t(8;21). In contrast, in a cohort of 43 AML patients, higher levels of LAT2 were associated with myelomonocytic features. Differentiation of HL-60 and NB4 cells towards granulocytes by all trans-retinoic acid (ATRA) resulted in downregulation of LAT2; conversely, it was upregulated during phorbol ester-induced monocytic differentiation of HL-60 cells. Forced expression of LAT2 in Kasumi-1 cells resulted in a striking block of ATRA- and phorbol ester-induced differentiation, implicating disturbances of the graded expression of this adaptor molecule in the maturation block of myeloid leukaemia cells.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Aged, 80 and over; Cell Differentiation; Cholecalciferol; Chromosomes, Human, Pair 21; Chromosomes, Human, Pair 8; Core Binding Factor Alpha 2 Subunit; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Granulocytes; Humans; Leukemia, Myeloid, Acute; Macrophages; Male; Middle Aged; Monocytes; Neoplasm Proteins; Oncogene Proteins, Fusion; Phorbol Esters; RUNX1 Translocation Partner 1 Protein; Translocation, Genetic; Tumor Cells, Cultured; Up-Regulation

Induction of terminal differentiation is a conceptually attractive approach for the therapy of neoplastic diseases. Although vitamin D derivatives (deltanoids) can induce differentiation of AML cells in vitro, so far deltanoids have not been successfully brought to the clinic, due to the likelihood of life-threatening hypercalcemia. Here, we incubated freshly obtained blood cells from patients with AML with a plant antioxidant (PAOx), silibinin (SIL), alone or together with a deltanoid. Twenty patients with AML (all subtypes except M3) were available for this study, and in 14 (70%), SIL (60 µM) either induced differentiation ex vivo, or enhanced differentiation induced by deltanoids, or both. Interestingly, SIL acting alone induced differentiation only in cases in which chromosome aberrations could not be detected. In eleven samples sufficient material was available for a limited analysis of the underlying events. Quantitative RT-PCR showed that differentiation markers were upregulated at the mRNA level by both SIL and deltanoids, suggesting that intracellular signaling pathways upstream of transcription factors (TFs) were activated by these agents. Western analysis for proteins which function as TFs in deltanoid-induced monocytic differentiation, such as members of Jun and C/EBP families, surprisingly demonstrated that SIL upregulated all these TFs in the cases tested. This suggests that although the presence of SIL may not always be sufficient to induce differentiation, it can serve as a differentiation enabling factor for blasts obtained from a large proportion of patients with AML. Thus, SIL/deltanoid combinations warrant further consideration as preventive/therapeutic regimens in human leukaemia.

Topics: Adult; Aged; Cell Differentiation; Cholecalciferol; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Monocytes; Receptors, Calcitriol; Retinoid X Receptor alpha; Reverse Transcriptase Polymerase Chain Reaction; Silybin; Silymarin; Transcription Factors; Up-Regulation

Acute leukemias are clonal disorders of hematopoiesis wherein a leukemic stem cell (LSC) acquires mutations that confer the capacity for unlimited self-renewal, impaired hematopoietic differentiation, and enhanced proliferation to the leukemic clone. Many recent advances in understanding the biology of leukemia have come from studies defining specific genetic and epigenetic abnormalities in leukemic cells. Three recent articles, however, further our understanding of leukemia biology by elucidating specific abnormalities in metabolic pathways in leukemic hematopoiesis. These studies potentially converge on the concept that modulation of reactive oxygen species (ROS) abundance may influence the pathogenesis and treatment of acute myeloid leukemia (AML).

Topics: Animals; Cholecalciferol; Glutarates; Hematopoiesis; Hematopoietic Stem Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Iron Chelating Agents; Isocitrate Dehydrogenase; JNK Mitogen-Activated Protein Kinases; Leukemia; Leukemia, Myeloid, Acute; Models, Biological; Mutation, Missense; NADP; Neoplastic Stem Cells; Reactive Oxygen Species

Differentiating agents have been proposed to overcome the impaired cellular differentiation in acute myeloid leukemia (AML). However, only the combinations of all-trans retinoic acid or arsenic trioxide with chemotherapy have been successful, and only in treating acute promyelocytic leukemia (also called AML3). We show that iron homeostasis is an effective target in the treatment of AML. Iron chelating therapy induces the differentiation of leukemia blasts and normal bone marrow precursors into monocytes/macrophages in a manner involving modulation of reactive oxygen species expression and the activation of mitogen-activated protein kinases (MAPKs). 30% of the genes most strongly induced by iron deprivation are also targeted by vitamin D3 (VD), a well known differentiating agent. Iron chelating agents induce expression and phosphorylation of the VD receptor (VDR), and iron deprivation and VD act synergistically. VD magnifies activation of MAPK JNK and the induction of VDR target genes. When used to treat one AML patient refractory to chemotherapy, the combination of iron-chelating agents and VD resulted in reversal of pancytopenia and in blast differentiation. We propose that iron availability modulates myeloid cell commitment and that targeting this cellular differentiation pathway together with conventional differentiating agents provides new therapeutic modalities for AML.

Topics: Aged; Animals; Antibodies, Monoclonal; Antigens, CD; Apoptosis; Blood Cell Count; CD11b Antigen; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cholecalciferol; Drug Synergism; Female; Gene Expression; Gene Expression Profiling; Granulocytes; Hematopoietic Stem Cells; Homeostasis; Humans; Hydroxycholecalciferols; Iron; Iron Chelating Agents; Iron Deficiencies; JNK Mitogen-Activated Protein Kinases; Kaplan-Meier Estimate; Leukemia, Myeloid, Acute; Lipopolysaccharide Receptors; Male; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Monocytes; Phosphorylation; Reactive Oxygen Species; Receptors, Calcitriol; Receptors, Transferrin; Xenograft Model Antitumor Assays

A number of reports indicate that peroxisome proliferator-activated receptor (PPAR) delta is involved in the molecular control of monocyte-macrophage differentiation. In this regard, the recent demonstration that PPARdelta is a primary response gene of 1alpha,25-dihydroxyvitamin D3 (VD), i.e. a powerful inducer of such process, allowed us to hypothesize the existence of a cross talk between PPARdelta and VD receptor pathways. To address this issue, we analyzed the effects promoted by stimulation with PPARdelta ligands and by overexpression of this nuclear receptor in monoblastic cell lines undergoing exposure to VD. The results obtained evidenced that, although promoting a weak differentiation effect by themselves, PPARdelta ligands efficiently co-operated with VD treatment. In spite of this, PPARdelta overexpression exerted a remarkable inhibitory effect on monocyte-macrophage differentiation induced by VD that was, at least partly, reverted by stimulation with a highly specific PPARdelta ligand. These data indicate that, although acting through a ligand-dependent modality, PPARdelta is a negative regulator of VD-mediated monocyte differentiation, allowing us to hypothesize a role of the investigated nuclear receptor in the differentiation block of M5 type (monoblastic) acute myeloid leukemias (AMLs). Bioinformatic analysis of a microarray database, containing the expression profiles of 285 AML cases, further supported this hypothesis demonstrating the existence of a subset of M5 type (monoblastic) AMLs that overexpress PPARdelta gene.

Topics: Antigens, CD34; Antigens, Differentiation; Arachidonic Acid; Cell Differentiation; Cell Line; Cholecalciferol; Computational Biology; Databases, Factual; Epoprostenol; Gene Expression Profiling; Hematopoiesis; Humans; Leukemia, Myeloid, Acute; Ligands; Monocytes; PPAR delta; Thiazoles; Up-Regulation

The majority of acute myeloid leukaemia (AML) patients are over sixty years of age. With current treatment regimens, survival rates amongst these, and also those younger patients who relapse, remain dismal and novel therapies are urgently required. In particular, therapies that have anti-leukaemic activity but that, unlike conventional chemotherapy, do not impair normal haemopoiesis.. Here we demonstrate the potent anti-leukaemic activity of the combination of the lipid-regulating drug bezafibrate (BEZ) and the sex hormone medroxyprogesterone acetate (MPA) against AML cell lines and primary AML cells. The combined activity of BEZ and MPA (B/M) converged upon the increased synthesis and reduced metabolism of prostaglandin D(2) (PGD(2)) resulting in elevated levels of the downstream highly bioactive, anti-neoplastic prostaglandin 15-deoxy Delta(12,14) PGJ(2) (15d-PGJ(2)). BEZ increased PGD(2) synthesis via the generation of reactive oxygen species (ROS) and activation of the lipid peroxidation pathway. MPA directed prostaglandin synthesis towards 15d-PGJ(2) by inhibiting the PGD(2) 11beta -ketoreductase activity of the aldo-keto reductase AKR1C3, which metabolises PGD(2) to 9alpha11beta-PGF(2alpha). B/M treatment resulted in growth arrest, apoptosis and cell differentiation in both AML cell lines and primary AML cells and these actions were recapitulated by treatment with 15d-PGJ(2). Importantly, the actions of B/M had little effect on the survival of normal adult myeloid progenitors.. Collectively our data demonstrate that B/M treatment of AML cells elevated ROS and delivered the anti-neoplastic actions of 15d-PGJ(2). These observations provide the mechanistic rationale for the redeployment of B/M in elderly and relapsed AML.

Topics: 3-Hydroxysteroid Dehydrogenases; Aldo-Keto Reductase Family 1 Member C3; Antigens, CD34; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bezafibrate; Cell Differentiation; Cell Line, Tumor; Cholecalciferol; Drug Screening Assays, Antitumor; Glutathione; Humans; Hydroxyprostaglandin Dehydrogenases; I-kappa B Proteins; Leukemia, Myeloid, Acute; Medroxyprogesterone Acetate; PPAR gamma; Prostaglandin D2; Reactive Oxygen Species; Vitamin A

Differentiation therapy with the hormonal form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1,25D(3)), is a promising approach to treatment of acute myeloid leukemia (AML); however, 1,25D(3) induces hypercalcemia at pharmacologically active doses. We investigated the in vitro and in vivoantileukemic efficacy of combined treatment with non-toxic doses of a low-calcemic 1,25D(3) analogue, 1,25-dihydroxy-21(3-hydroxy-3-methyl-butyl)-19-nor-cholecalciferol (19-nor-Gemini; Ro27-5646), and rosemary plant agents in a mouse model of AML.. Proliferation and differentiation of WEHI-3B D- (WEHI) murine myelomonocytic leukemia cellsin vitro were determined by standard assays. Reactive oxygen species, glutathione and protein expression levels were measured by flow cytometry, enzymatic assay and Western blotting, respectively. Systemic AML was developed by intravenous injection of WEHI cells in syngeneic Balb/c mice.. 19-nor-Gemini had a higher potency than its parent compounds, Gemini (Ro27-2310) and 1,25D(3), in the induction of differentiation (EC(50) = 0.059 +/- 0.011, 0.275 +/- 0.093 and 0.652 +/- 0.085 nM, respectively) and growth arrest (IC(50) = 0.072 +/- 0.018, 0.165 +/- 0.061 and 0.895 +/- 0.144 nM, respectively) in WEHI cells in vitro, and lower in vivo toxicity. Combined treatment of leukemia-bearing mice with 19-nor-Gemini (injected intraperitoneally) and standardized rosemary extract (mixed with food) resulted in a synergistic increase in survival (from 42.2 +/- 2.5 days in untreated mice to 66.5 +/- 4.2 days, n = 3) and normalization of white blood cell and differential counts. This was consistent with strong cooperative antiproliferative and differentiation effects of low concentrations of 19-nor-Gemini or 1,25D(3) combined with rosemary extract or its major polyphenolic component, carnosic acid, as well as with the antioxidant action of rosemary agents and vitamin D derivatives in WEHI cell cultures.. Combined effectiveness of 1,25D(3) analogues and rosemary agents against mouse AML warrants further exploration of this therapeutic approach in translational models of human leukemia.

Topics: Abietanes; Animals; Antioxidants; Bone Marrow; Calcitriol; Cell Differentiation; Cell Proliferation; Cholecalciferol; Disease Models, Animal; Drug Synergism; Humans; Immunoblotting; Leukemia, Experimental; Leukemia, Myeloid, Acute; Mice; Mice, Inbred BALB C; Plant Extracts; Reactive Oxygen Species; Rosmarinus; Survival Rate; Tumor Cells, Cultured

The innate immune system of mammals provides a rapid response to repel assaults from numerous infectious agents including bacteria, viruses, fungi, and parasites. A major component of this system is a diverse combination of cationic antimicrobial peptides that include the alpha- and beta-defensins and cathelicidins. In this study, we show that 1,25-dihydroxyvitamin D3 and three of its analogs induced expression of the human cathelicidin antimicrobial peptide (CAMP) gene. This induction was observed in acute myeloid leukemia (AML), immortalized keratinocyte, and colon cancer cell lines, as well as normal human bone marrow (BM) -derived macrophages and fresh BM cells from two normal individuals and one AML patient. The induction occurred via a consensus vitamin D response element (VDRE) in the CAMP promoter that was bound by the vitamin D receptor (VDR). Induction of CAMP in murine cells was not observed and expression of CAMP mRNA in murine VDR-deficient bone marrow was similar to wild-type levels. Comparison of mammalian genomes revealed evolutionary conservation of the VDRE in a short interspersed nuclear element or SINE in the CAMP promoter of primates that was absent in the mouse, rat, and canine genomes. Our findings reveal a novel activity of 1,25-dihydroxyvitamin D3 and the VDR in regulation of primate innate immunity.

Topics: Antimicrobial Cationic Peptides; Bone Marrow Cells; Calcitriol; Cathelicidins; Cell Differentiation; Cell Line, Tumor; Cholecalciferol; Cytokines; Evolution, Molecular; Gene Expression Regulation; Humans; Leukemia, Myeloid, Acute; Monocytes; Promoter Regions, Genetic; Receptors, Calcitriol; Response Elements; RNA, Messenger

We examined telomerase activity in myeloid leukaemic cell lines, normal haemopoietic cells, and leukaemic blasts from acute myelogenous leukaemia (AML) patients. Normal bone marrow mononuclear (BMNC) cells expressed low telomerase activity. Higher telomerase activity was detected in 10 myeloid leukaemic cell lines compared to normal BMNC cells. Treatment with 1,25(OH)2D3, and vitamin D3 analogues, EB1089 and KH1060, reduced telomerase activity in vitamin D3-sensitive HL-60 cells, whereas vitamin D3 insensitive K562 cells did not change its activity. This down-regulation of telomerase activity by EB1089 was associated with induction of p21 protein. The rank order of telomerase activity was leukaemic CD34- cells > leukaemic CD34+ cells > normal CD34- cells > normal CD34+ cells. Telomerase activity was positive in all of the AML patients tested; however, heterogeneity of telomerase activity was found amongst this group. Therefore we compared telomerase activity with clinical response. Unexpectedly, we found that a higher rate of complete remission was noted in AML patients with higher telomerase activity. No association between telomerase activity and biological parameters including percentage of S-phase, cytotoxicity to cytosine arabinoside and percentage of CD34+ cells in AML blasts was found. These results suggest that telomerase activity in AML patients is detected with high frequency, but is heterogenous. Expression level of telomerase activity may have a clinical implication in AML patients regarding clinical response.

Topics: Adult; Aged; Antigens, CD34; Cholecalciferol; Female; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Telomerase; Tumor Cells, Cultured

Two homologous high-affinity receptors for the chemoattractant interleukin-8, IL-8RA and IL-8RB, and one for the chemoattractant C5a (C5aR) have been cloned. These membrane proteins are members of the rhodopsin superfamily of G-protein coupled seven-transmembrane segment receptors. New monoclonal antibodies (mAb) directed against the deduced N-terminal sequences of the IL-8RA (mAb SE2) and IL-8RB (mAb HC2) were generated to determine the IL-8R expression on human blood leukocytes and two human myeloid cell lines. The C5aR expression was detected using the mAb W17/1. Approximately 107,000 C5aR, 55,000 IL-8RA, and 25,000 IL-8RB molecules per cell could be detected on human granulocytes by flow cytometric analysis. On peripheral blood monocytes, 42,000 C5aR molecules/cell and 3000 IL-8RB molecules/cell were expressed. However, we were unable to quantitate IL-8RA expression, which was detectable but below 2500 molecules per cell and thus outside the standard range for the quantitation of receptor molecules by flow cytometry. On AML-193 cells, only the IL-8RB was constitutively expressed, whereas on HL-60 cells, we could not detect expression of any of the three receptors. Vitamin D3 (250 ng/ml, 7 days), which has been shown to induce differentiation of AML-193 and HL-60 cells into the monocytic phenotype, led to an up-regulation of IL-8RB and C5aR in both cell lines in the absence of any expression of IL-8RA. In contrast, all-trans retinoic acid (0.1 microM, 7 days), which induces differentiation into the granulocytic phenotype, led to an up-regulation of IL-8RB in AML-193 cells and to an expression of IL-8RB and C5aR in HL-60 cells. Again, neither cell line expressed IL-8RA. These findings suggest that regulation of IL-8RA expression differs from that of its IL-8RB homolog and may be a late event in leukocyte maturation.

Topics: Antigens, CD; Cell Differentiation; Chemotactic Factors; Cholecalciferol; Complement C5a; GTP-Binding Proteins; HL-60 Cells; Humans; Interleukin-8; Leukemia, Myeloid, Acute; Leukocytes; Protein Binding; Receptor, Anaphylatoxin C5a; Receptors, Complement; Receptors, Interleukin; Receptors, Interleukin-8A; Tretinoin; Virulence Factors, Bordetella

Bryostatin 5 is a macrocyclic lactone which activates protein kinase C (PKC). PKC activation has been implicated in leukemic cell differentiation. We have examined the effect of PKC activation by bryostatin 5 on human acute myeloid cell differentiation in the presence and absence of vitamin D3. In vitro treatment of 20 patient samples of acute myeloid leukemias in a 4 days culture system with 10 nM bryostatin 5 induced strongly adherent macrophage-like cells in all cases. Bryostatin 5 induced a significant (p = 0.00006) increment in esterase activity in a majority of the samples, which was further enhanced by vitamin D3. CD14 expression was significantly (p = 0.035) enhanced with the combination of bryostatin 5 and vitamin D3. Nitroblue tetrazolium (NBT) reducing ability was, however, nearly abolished (p = 0.0007). A loss of CD34 expression occurred during cell culture; this loss was enhanced by vitamin D3, but prevented partly by bryostatin 5. Together these findings indicate that exposure to bryostatin 5 leads to a strong macrophage-like cell differentiation in human myeloid leukemia and that VD3 has an additional effect. These findings strengthen the potential role of bryostatins as possible antileukemic agents.

Topics: Adolescent; Adult; Aged; Antigens, CD; Antigens, CD34; Antigens, Differentiation, Myelomonocytic; Antineoplastic Agents; Bryostatins; Carboxylic Ester Hydrolases; Cell Differentiation; Cholecalciferol; Drug Synergism; Enzyme Activation; Female; Humans; Lactones; Leukemia, Myeloid, Acute; Lipopolysaccharide Receptors; Macrolides; Macrophages; Male; Middle Aged; Protein Kinase C; Tumor Cells, Cultured

A patient with myelodysplastic syndrome (MDS) and a 47,XY,+21 karyotype at diagnosis, was documented to have a clonal chromosome 21 rearrangement, i(21q), four months before transformation to acute biphenotypic leukemia. For 4 months after transformation, isochromosome 21 persisted while the patient was receiving treatment with zidovudine. Vitamin D3 was added to zidovudine for an additional month, during which time the trisomy 21 clone reappeared as the predominant cell population. The unique aspects of this patient are the atypical evolution of chromosome 21, the transformation to biphenotypic leukemia, and the occurrence of i(21q) associated with biphenotypic leukemia evolving from an MDS.

Topics: Anemia, Refractory, with Excess of Blasts; Antigens, CD; Antigens, Differentiation, Myelomonocytic; CD13 Antigens; Cell Transformation, Neoplastic; Cholecalciferol; Chromosomes, Human, Pair 21; Gene Expression Regulation, Leukemic; Gene Rearrangement; Humans; Immunophenotyping; Leukemia, Myeloid, Acute; Receptors, Interleukin-2; Trisomy; Zidovudine

We studied differentiation inducing effects of retinoic acid (RA), 1 alpha, 25-dihydroxyvitamin D3 (D3) and interferons (IFNs), alone and in combination, on fresh myeloid leukemic cells from 8 patients. RA not only induced the differentiation of leukemic cells in 5/8 cases, but potentiated differentiation by IFNs either in granulocytic or monocytic pathways. In particular, interferon-alpha enhanced granulocytic differentiation and interferon-gamma induced mono-macrophage differentiation of promyelocytic leukemic cells in the presence of RA. Differentiation induced by D3, alone or in combination with IFNs, was limited in all cases. RA plus IFNs might be an effective combination for differentiation therapy for some types of myeloid leukemia.

Topics: Cell Differentiation; Cholecalciferol; Esterases; Humans; Interferon Type I; Interferon-gamma; Leukemia, Myeloid, Acute; Muramidase; Nitroblue Tetrazolium; Tretinoin; Tumor Cells, Cultured

A 57-year-old woman was admitted to our hospital because of further examination of anemia in November, 1978. She was diagnosed as having aplastic anemia, which was not associated with any atypical findings. Treatment with oxymetholone had been effective. However, she developed pancytopenia in January, 1988. A bone marrow aspiration revealed a hypocellular marrow with 75.6% blasts. Following two courses with low dose cytosine arabinoside and vitamin D3, the leukemia improved although she needs red cell transfusions at intervals of one to two months because of persistent pancytopenia. We present a patient with aplastic anemia who developed a hypoplastic leukemia at the tenth year of the disease.

Topics: Anemia, Aplastic; Bone Marrow; Cholecalciferol; Cytarabine; Female; Humans; Leukemia, Myeloid, Acute; Middle Aged

From ten consecutive patients with acute myeloid leukemia leukemic cells were isolated and cultured with and without 10(-6), 10(-7) and 10(-8) M 1.25(OH)2D3 (vit D3), retinoic acid (RA) cytosine-arabinoside (ARA-C) and 1.0, 1.25 and 1.5% dimethyl sulfoxide (DMSO). Maturation was measured with a comprehensive panel of qualitative and quantitative parameters of maturation. Six of those ten leukemias showed significant (p less than 0.01) changes in at least three parameters after exposure to either one of the differentiation inducers. Vit D3 induced maturation in four leukemias, in three of them clearly in monocytic direction. ARA-C showed changes in one leukemia in only three parameters not pointing to either granulocytic or monocytic direction. Maturation in granulocytic direction was observed after exposure to RA in one leukemia. Maturation induction was observed in six out of ten freshly isolated leukemic cells with vit D3 being the most potent inducer of maturation in monocytic direction. The data about inducibility of maturation in freshly isolated human leukemic cells are reviewed and discussed.

Topics: Adult; Aged; Cell Differentiation; Cell Separation; Cholecalciferol; Cytarabine; Dimethyl Sulfoxide; Female; Granulocytes; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Monocytes; Neoplastic Stem Cells; Tretinoin; Tumor Cells, Cultured

It is known that 1 alpha, 25(OH)2D3 is an inducer of nonlymphoid leukemic cell differentiation to monocyte-macrophage-like in vitro. The effects of oral administration of 4.5-15 micrograms/d 1 alpha (OH)D3, which is converted to 1 alpha, 25(OH)2D3 in vivo by liver cells, on leukemic cells were studied in two patients with AML and one with RAEB. In these three cases, 1 alpha (OH)D3 reduced the number of leukemic cells in the bone marrow and aggregated the dispersed chromatin of leukemic cells as heterochromatin. Furthermore, this drug induced atypical lymphocyte-like cells, which were considered to be differentiated from leukemic cells in case 1, and improvement of pancytopenia in case 3. While hypercalcemia developed during 1 alpha (OH)D3 therapy in case 1, it disappeared within three days after discontinuation. We also studied the in vitro effects of 1 alpha, 25 (OH)2D3 on leukemic cells freshly isolated from the bone marrow in these three cases. After incubation with 10(-8) M 1 alpha, 25(OH)2D3 at 37 degrees C for 72 h, the number of adherent cells on the bottom of Petri dishes had increased. These cells were quite similar to monocyte-macrophages. These leukemic cells, after differentiation induced by 1 alpha, 25(OH)2D3, reacted strongly with monoclonal antibodies My-4 and My-7. Both 100 microM D-cis and L-cis diltiazem (calcium channel blocker) enhanced the differentiation of HL-60 cells induced by 1 alpha, 25 (OH)2D3. There was no significant difference between D-cis and L-cis diltiazem with regard to this enhancing effect. The cytoplasmic free Ca2+ concentration in HL-60 cells induced by 1 alpha, 25(OH)2 D3 and/or diltiazem, was increased significantly as compared with that in HL-60 cells incubated without inducers.

Topics: Administration, Oral; Aged; Anemia, Refractory, with Excess of Blasts; Calcium; Cell Differentiation; Cholecalciferol; Diltiazem; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Myelodysplastic Syndromes

Topics: Aged; Bone Marrow; Child; Cholecalciferol; Combined Modality Therapy; Cytarabine; Female; Follow-Up Studies; Humans; Interferon Type I; Leukemia, Myeloid, Acute; Male; Middle Aged; Prognosis; Tretinoin

Human monocytic tumor cells of the U937 cell line contain substantial quantities of two neutrophil neutral proteinases, elastase and cathepsin G, raising the question of whether their presence reflects an expression of transformation or whether normal monocytes undergo a developmental stage in which they produce certain neutrophil proteinases. To address this issue, we examined U937 cells for production of collagenase, since human alveolar macrophages release fibroblast-like collagenase, an enzyme that is distinct from neutrophil collagenase. Using an immunoassay that utilized antibody to skin fibroblast collagenase, we found that U937 cells secreted barely detectable quantities of enzyme, 10-12 ng/10(6) cells per 24 h, under basal conditions. Upon incubation with 10 nM 12-o-tetradecanoyl-phorbol-13-acetate (TPA), however, collagenase release increased 200-fold, comparable to the amount secreted by phorbol-stimulated human fibroblasts. Metabolic labeling and immunoprecipitation confirmed the enhanced synthesis of U937 cell collagenase upon TPA exposure. This enzyme activity further resembled fibroblast collagenase and differed from neutrophil collagenase by exhibiting preferential cleavage of monomeric type III collagen relative to type I. As previously observed with human alveolar macrophages, U937 cells also released a protein identical to the collagenase inhibitor produced by human skin fibroblasts, a molecule not associated with neutrophils. Release of this inhibitor increased 10-fold with TPA exposure. In contrast to collagenase and collagense inhibitor, TPA-treated U937 cells contained only 10-15% as much elastase and cathepsin G activities as control cells. Thus, TPA-induced differentiation modified the presence of these enzymes in the direction of their content in normal monocytes. Since the neutral proteinase profile of undifferentiated U937 cells resembles that of neutrophils and changes markedly after cellular differentiation to one that is characteristic of monocytes, these data suggest that neutrophilic proteinases may be produced by normal monocytes during the early stages of their differentiation.

Topics: Cathepsin G; Cathepsins; Cell Differentiation; Cell Line; Chemotaxis; Cholecalciferol; Cyclic AMP; DNA; Humans; Leukemia, Myeloid, Acute; Macrophages; Microbial Collagenase; Monocytes; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pancreatic Elastase; Phorbols; Serine Endopeptidases; Tetradecanoylphorbol Acetate

Monocytic differentiation of cultured leukemic cells HL60 can be induced by a variety of compounds including analogs of vitamin D3. We studied the expression of oncogenes c-fos, c-fms, c-fes, c-myb, c-myc, and c-Ha-ras during this process, and attempted to relate these and a conventional marker of monocytic differentiation, the nonspecific esterase activity, to changes in cytosol levels of the Ca2+-binding proteins. The analogs of vitamin D3, 1 alpha,25-dihydroxycholecalciferol, 1 alpha,25-dihydroxy-26,27-hexafluorocholecalciferol, and 1 alpha,25-dihydroxy-24R-fluorocholecalciferol, reduced the total calcium binding activity and the cellular levels of calmodulin, but calbindin D28k could not be detected in HL 60 cells. A correlation was evident between the potency of these compounds as inducers of differentiation demonstrated by nonspecific esterase activity, the degree of reduction in calmodulin concentration, the rate of the inhibition of DNA synthesis and of expression of c-myc and c-myb genes, and the induction of the expression of oncogenes c-fos and c-fms. These experiments indicate that the events which underlie monocytic differentiation of HL 60 cells can be observed to occur in discrete steps which include an inhibition of DNA synthesis, a reduction of cellular levels of calmodulin, and altered expression of several oncogenes.

Topics: Annexin A6; Calcitriol; Calcium-Binding Proteins; Calmodulin; Cell Differentiation; Cell Line; Cholecalciferol; DNA Replication; Gene Expression Regulation; Humans; Leukemia, Myeloid, Acute; Monocytes; Nucleic Acid Hybridization; RNA, Messenger

The human promyelocytic leukemia cell line HL-60 undergoes macrophage-like differentiation after exposure to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D3. In the current study, we demonstrate that 1,25(OH)2D3 also regulates 25-hydroxyvitamin D3 [25(OH)D3] metabolism in HL-60 cells. The presence of 1,25(OH)2D3 in the culture medium of HL-60 cells stimulated the conversion of 7-10% of the substrate [25(OH)D3] to a more polar metabolite, which was identified as 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] from the elution positions on sequential HPLC systems and the sensitivity to periodate treatment. The HL-60 subclone HL-60 blast, which is unresponsive to 1,25(OH)2D3 in terms of differentiation, also responded to 1,25(OH)2D3 treatment with the production of 24,25(OH)2D3. Maximal stimulation of 24,25(OH)2D3-synthesis (approximately 7 pmol/5 X 10(6) cells) in HL-60 cells was noted with a 12-h exposure to 10(-9) M 1,25(OH)2D3. The ability of vitamin D3 metabolites other than 1,25(OH)2D3 to induce the synthesis of 24,25(OH)2D3 in HL-60 cells was, with the exception of 1 alpha-hydroxyvitamin D3, in correlation with their reported affinities for the specific 1,25(OH)2D3 receptor which is present in HL-60 cells. Treatment of HL-60 cells with phorbol diesters abolished the 1,25(OH)2D3 responsiveness, while treatment with dimethylsulfoxide and interferon gamma did not markedly alter the 25(OH)D3 metabolism of HL-60 cells. Small amounts (approximately 1% of substrate) of two 25(OH)D3 metabolites, which comigrated with 5(E)- and 5(Z)-19-nor-10-keto-25-hydroxyvitamin D3 on two HPLC solvent systems, were synthesized by HL-60 cells, independently from 1,25(OH)2D3 treatment or stage of cell differentiation. Our results indicate that 1,25(OH)2D3 influences 25(OH)D3 metabolism of HL-60 cells independently from its effects on cell differentiation.

Topics: Calcitriol; Cell Differentiation; Cell Line; Cholecalciferol; Dihydroxycholecalciferols; Humans; Leukemia, Myeloid, Acute; Macrophages; Periodic Acid; Tetradecanoylphorbol Acetate

A leukemic cell line (HL60) and acute myeloblastic leukemia (AML) cells from six patients were co-cultured with normal marrow cells to assess their effects on growth of normal CFU-GEMM. The effects of the following inducers: 12-0-tetradecanoyl-phorbol-13-acetate (TPA), retinoic acid (RA), dimethylsulphoxide (DMSO), 1-25 (OH) D3 (Vitamin D3) and PHA-LCM on both the HL60 and AML cells, were studied. Inhibition of growth of normal CFU-GEMM was observed in the co-cultures in the presence of 1 X 10(4) HL60 or AML leukemic cells/ml. This inhibition was reversed by pretreating the HL60 line with vitamin D3, TPA and RA. No effect on growth of CFU-GEMM was noted when DMSO and PHA-LCM were used. AML cells were morphologically induced to differentiate by TPA or RA in all six cases. In three cases, reversal of inhibition of growth of normal pluripotent hemopoietic progenitors occurred and in three the inhibition of growth persisted. Regulation of inhibition by different inducers did not seem to correlate in all cases with morphological differentiation.

Topics: Cell Differentiation; Cell Line; Cholecalciferol; Culture Techniques; Dimethyl Sulfoxide; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid, Acute; Leukocyte Count; Neoplastic Stem Cells; Phytohemagglutinins; Stem Cells; Tetradecanoylphorbol Acetate; Tretinoin

We studied the effects of 1,25-dihydroxyvitamin D3 and other metabolites of vitamin D3 on the maturation in liquid culture and on colony formation in semisolid media of marrow and buffy coat cells from patients with myeloid leukemias and from normal individuals. After incubation with 1,25-dihydroxy-vitamin D3, a proportion of both normal and leukemic myeloid cells resembled cells of the monocyte-macrophage lineage; these cells expressed alpha-naphthylacetate esterase and were able to phagocytize and kill candida organisms. When granulocyte-macrophage progenitor cells (CFU-GM) were incubated with 1,25-dihydroxyvitamin D3, the number of monocyte-macrophage colonies was increased and the number of granulocyte colonies was reduced; megakaryocyte colony formation (CFU-Mk) was inhibited substantially; and there was no effect on erythroid (BFU-E) or multilineage (CFU-GEMM) progenitor cell colony formation. We propose that 1,25-dihydroxyvitamin D3 may induce cells that are normally committed to differentiate along the granulocytic pathways to differentiate instead along the monocyte-macrophage pathway. If these in vitro observations reflect the in vivo activity of 1,25-dihydroxyvitamin D3, it may be involved in the modulation of collagen deposits in the bone marrow.

Topics: 24,25-Dihydroxyvitamin D 3; Bone Marrow; Calcifediol; Calcitriol; Cell Division; Cells, Cultured; Cholecalciferol; Dihydroxycholecalciferols; Granulocytes; Hematopoietic Stem Cells; Humans; Kinetics; Leukemia, Myeloid, Acute; Macrophages

Induction of cell differentiation by twenty-one derivatives of vitamin D3 and their binding affinity for the receptor of 1 alpha,25-dihydroxyvitamin D3, an active vitamin D3 metabolite, were examined using HL-60 human promyelocytic leukemia cells and chick intestinal cytosol, respectively. 1 alpha,24(R)-dihydroxy-26-chlorovitamin D3 is found to be more active than 1 alpha,24(R)-dihydroxyvitamin D3 and 1 alpha,25-dihydroxyvitamin D3 in the two abilities to induce the differentiation and to bind to the receptor. The results suggest that a hydroxy group at C-1 position of vitamin D3 and a hydroxy or oxo group at C-25 or C-24 position are essential for both abilities. In diastereoisomers of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone, synthesized (23 S,25 S) 1 alpha,25-dihydroxyvitamin D3-26,23-lactone was more active in both abilities than the natural metabolite, (23 S,25 R) 1 alpha,25-dihydroxyvitamin D3-26,23-lactone, which shows anti-vitamin D action.

Topics: Animals; Cell Differentiation; Cell Line; Chickens; Cholecalciferol; Cytosol; Humans; In Vitro Techniques; Intestinal Mucosa; Leukemia, Myeloid, Acute; Nitroblue Tetrazolium; Receptors, Calcitriol; Receptors, Steroid; Stereoisomerism; Vitamin D Deficiency