Greater synergism of retinoic acid receptor (RAR) agonists with vitamin D3 than that of retinoid X receptor (RXR) agonists with regard to growth inhibition and differentiation induction in monoblastic leukemia cells

Retinoids and 1alpha,25-dihydroxyvitamin D3 (VD3) cooperatively induce the differentiation of myeloid leukemia cells. We investigated the role of retinoid receptors (RARs and RXRs) in the combined effects of retinoids and VD3 on growth inhibition and differentiation induction in human monoblastic leukemia U937 cells by using RAR- or RXR-selective retinoids. An isobologram analysis showed that both combinations were synergistic with regard to inhibiting the proliferation, and RAR agonists exhibited greater synergism with VD3 than did RXR agonists. RXR agonists alone induced nitroblue tetrazolium (NBT) reduction and expression of CD11b in U937 cells, whereas RAR agonists alone did not. On the other hand, RAR agonists and RXR agonists enhanced the differentiation induced by VD3, but RXR agonists required higher concentrations. An RAR antagonist inhibited the differentiation induced by RAR agonists plus VD3, but not that induced by RXR agonists plus VD3. Thus, RARs and RXRs act differently in their synergism with VD3. RAR agonists are more potent than RXR agonists with regard to synergism with VD3, and their combination may be useful in differentiation therapy against myeloid leukemia.     

Duplication-based measures of difference between gene and species trees

HL60 cells differentiate to monocytes or neutrophils in response to 1 alpha,25(OH)2-vitamin D3 (D3) and retinoids respectively. D3 and retinoid actions converge since their receptors (VDR, RAR) heterodimerise with a common partner, RXR, which also interacts with thyroid hormone (T3) receptors (T3R). HL60 cells were treated with combinations of D3 and retinoids to induce differentiation and to investigate whether increased VDR or RAR expression correlated with monocyte or neutrophil differentiation and whether altered receptor concentrations affected DNA-binding specificity. As assessed by Western blotting, VDR and RXR expression was unchanged in monocytes relative to controls but levels of RAR and T3R were reduced. In contrast, only VDR expression was reduced in neutrophils. T3 did not promote differentiation or influence its induction by D3 or retinoids and did not affect expression of any receptor. Gel mobility-shift analysis revealed that nuclear extracts from undifferentiated cells, monocytes and neutrophils interacted differently with VDRE-, RARE- and RXRE-binding sites. Monocyte nuclear protein/DNA complexes contain readily detectable VDR and RXR whereas neutrophil complexes contain RAR and RXR. Thus hormone-induced changes in receptor stoichiometry favour either VDR/RXR or RAR/RXR heterodimerisation and correlate with hormone-induced differentiation of HL60 cells to monocytes or neutrophils respectively.     

The histostructure of the adrenal gland in the Campbell hamster after extirpation of a specific midventral gland]

The receptors for retinoic acid (RA) and for 1 alpha,25-dihydroxyvitamin D3 (VD), RAR, RXR, and VDR are ligand-inducible members of the nuclear receptor superfamily. These receptors mediate their regulatory effects by binding as dimeric complexes to response elements located in regulatory regions of hormone target genes. Sequence scanning of the tumor necrosis factor-alpha type 1 receptor (TNF alpha RI) gene identified a 3' enhancer region composed of two directly repeated hexameric core motifs spaced by 2 nucleotides (DR2). On this novel DR2-type sequence, but not on a DR5-type RA response element, VD was shown to act through its receptor, the vitamin D receptor (VDR), as a repressor of retinoid signalling. The repression appears to be mediated by competitive protein-protein interactions between VDR, RAR, RXR, and possibly their cofactors. This VDR-mediated transrepression of retinoid signaling suggests a novel mechanism for the complex regulatory interaction between retinoids and VD.     

Induction of differentiation of myelogenous leukemia cells by humulone, a bitter in the hop

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D3 (VD3), inhibits proliferation and induces differentiation of myelomonocytic leukemia cells, but its clinical use is limited by the adverse effect of hypercalcemia. VD3 mobilizes calcium stores from bone by inducing the dissolution of bone mineral and matrix. We have recently found that humulone, a bitter in the hop extract for beer brewing, effectively inhibits bone resorption. In this study we examined the effect of humulone on the differentiation of human myelogenous leukemia cells. Humulone alone inhibited the growth of monoblastic leukemia U937 cells while only slightly increasing differentiation markers such as nitroblue tetrazolium (NBT)-reducing and lysozyme activities. Humulone effectively enhanced the differentiation-inducing action of VD3. Other myelomonocytic leukemia cells were induced to differentiate by VD3 and this was also enhanced by humulone. Since humulone is a less-toxic inhibitor of bone resorption, the combination of humulone and VD3 may be useful in differentiation therapy of myelomonocytic leukemia.     

Suppression of ANP gene transcription by liganded vitamin D receptor: involvement of specific receptor domains

We showed previously that liganded vitamin D receptor (VDR) effects a suppression of human atrial natriuretic peptide (hANP) gene-promoter activity in cultured neonatal rat atrial myocytes. In the present study, we have attempted to identify the structural domains of the VDR that are involved in mediating this suppression. We examined the effects of a series of VDR mutants on a cotransfected hANP promoter-driven chloramphenicol acetyltransferase (CAT) reporter. Neither the native VDR nor any of the mutants tested displayed inhibitory activity in the absence of the 1,25-dihydroxyvitamin D3 (VD3) ligand. Delta134, a deletant harboring solely the DNA binding region of the VDR, and L254G, a mutant shown to be defective in retinoid X receptor (RXR) heterodimer formation in other systems, were as effective as the native VDR in reducing promoter activity. HBD, a deletant containing only the hormone-binding domain of the VDR, and K246G, a point mutant that is defective in the activation function of the receptor, did not attenuate reporter activity. A similar activity profile was displayed when a positively regulated promoter containing a direct-repeat vitamin D responsive element (DR3-CAT) was examined in these cells. Liganded VDR, the delta134 mutant, and liganded L254G effected increases in DR3-CAT activity of 2.5-, 2-, and 4-fold, respectively. Two nonhypercalcemic analogues of VD3 (RO 23-7553 and RO 25-6760) displayed the same inhibitory activity as VD3. These studies suggest that the inhibition of hANP promoter activity requires both the DNA binding and activation functions of the receptor but does not appear to require formation of a classic RXR alpha-VDR heterodimer.     

Neplanocin A, a potent inhibitor of S-adenosylhomocysteine hydrolase, potentiates granulocytic differentiation of acute promyelocytic leukemia cells induced by all-trans retinoic acid

Several neplanocin A analogs were synthesized and their growth-inhibiting and differentiation-inducing activities on myelogenous leukemia cells were examined. An adenosine kinase-ineffective analog of neplanocin A was effective in inducing differentiation, suggesting that phosphorylation of the nucleoside is not essential for inducing the differentiation of leukemia cells. Neplanocin A induced functional and morphological differentiation of HL-60 cells, but did not effectively induce differentiation of NB4, a cell line derived from a leukemia patient with t(15;17). However, these cells have been known to undergo granulocytic differentiation upon treatment with all-trans retinoic acid (ATRA), and are used as a model for differentiation therapy in acute promyelocytic leukemia. Preexposure of NB4 cells to low concentrations of neplanocin A greatly enhanced the ATRA-induced differentiation of the cells, whereas representative antileukemic drugs such as cytosine arabinoside and daunomycin did not enhance this differentiation. A clinical strategy that combines intermittent treatment with neplanocin A analogs and a low dose of ATRA may increase the clinical response and decrease the adverse effects of ATRA.     

Expression of NADPH oxidase is induced by all-trans retinoic acid but not by phorbol myristate acetate and 1,25 dihydroxyvitamin D3 in the human promyelocytic cell line NB4

Human promyelocytic cells, NB4, differentiate into neutrophils in response to all-trans retinoic acid (ATRA). It has recently been proposed that NB4 cells have bilineage potential because these cells are also able to differentiate into monocyte/macrophages when exposed to a combination of 1,25-dihydroxyvitamin D3 (VD3) and phorbol myristate acetate (PMA). Differentiation of myeloid cells into neutrophils or monocytes is associated with the acquisition of the O2- producing enzyme, NADPH oxidase, which plays a critical role in microbial killing. In this study, the expression of the components of the NADPH oxidase complex during the differentiation of NB4 cells into neutrophils or macrophages has been investigated. Whereas cells exposed to ATRA were able to produce O2- after 2 days of differentiation, they remain unable to generate O2- when exposed to PMA or PMA + VD3. With the exception of p21rac, none of the other oxidase components was expressed in non-differentiated cells. Addition of ATRA induced the progressive expression and accumulation of p22phox, p91phox, p47phox and p67phox. Compared to the other components, p67phox was expressed late and its expression appeared to correlate most closely with the generation of O2- in the differentiation process. In PMA or PMA + VD3-differentiated NB4 cells, expression of the NADPH oxidase components was incomplete. Therefore, ATRA induced the expression of a functional NADPH oxidase complex in neutrophil-like NB4 cells. In contrast, when NB4 cells are exposed to monocytic differentiating agents, they acquire only part of the phenotypic characteristics of monocytes and lack one of the major phagocytic functionalities, the respiratory burst oxidase.     

Effect of acute acipimox administration on the rates of lipid and glycogen synthesis in cachectic tumor-bearing rats

2'-Deoxycoformycin (dCF), a specific and potent inhibitor of adenosine deaminase, has demonstrated significant antitumor effect on lymphoid malignancies. The drug induced functional and morphologic differentiation of myeloid leukemia cells in combination with 2'-deoxyadenosine (dAd), but not dCF alone. NB4, a cell line derived from a patient with t(15; 17) acute promyelocytic leukemia (APL) underwent granulocytic differentiation when treated with all-trans retinoic acid (ATRA) or dCF plus dAd, but not with cytosine arabinoside. Pre-exposure of NB4 cells to ATRA greatly potentiated differentiation induced by dCF plus dAd, but pretreatment with dCF plus dAd before exposure to ATRA was less effective. Differentiation of NB4 cells was effectively induced by clinically applicable concentrations of dCF in combination with dAd. These findings may provide useful information about induction of differentiation in vivo.     

Effects of prostaglandin E2 and all-trans retinoic acid combination on induced differentiation of human acute promyelocytic leukemia NB4 cells

OBJECTIVE: A human promyelocytic leukemia (APL) cell line NB4 was used to demonstrate the synergistic effects between all-trans retinoic acid (ATRA) and prostaglandin E2 (PGE2) on growth inhibition and cytodifferentiation induction. METHODS: NB4 cells were cultured in the presence of either ATRA or PGE2 as a single agent or in combinations at various ratio. Cell growth was measured and myeloid differentiation was tested on consecutive days over the whole course of culture. RESULTS: PGE2 and ATRA synergistically induced the myeloid differentiation of NB4 cells. In comparison with ATRA alone, the combination of PGE2 with ATRA caused an almost 20-fold decrease of effective concentration of ATRA. CONCLUSION: The combination of ATRA and PGE2 at an appropriate ratio may provide a convenient oral regimen of combined differentiation therapy for a better clinical outcome in APL patients.     

Disruption of vitamin D receptor-retinoid X receptor heterodimer formation following ras transformation of human keratinocytes

A partial resistance to the growth inhibitory influence of 1, 25-dihydroxyvitamin D3 is apparent when immortalized keratinocytes are transformed by the ras oncogene. The vitamin D receptor (VDR) was isolated, analyzed, and found to be identical in normal, immortalized, and ras-transformed keratinocytes. Subsequently, nuclear extracts from immortalized and ras-transformed keratinocytes were analyzed in gel mobility shift assays utilizing labeled vitamin D response elements or thyroid hormone response elements. A specific protein.DNA complex that was shown to contain VDR using an anti-VDR antibody was identified in both types of extracts; however, the addition of an anti-retinoid X receptor (RXR) antibody identified RXR in the complex of both normal and immortalized keratinocyte cell extracts, but not in ras-transformed keratinocytes. Furthermore, transfection of ras-transformed keratinocytes with wild-type human RXRalpha rescued VDR.RXR and thyroid hormone receptor.RXR complexes as demonstrated by a supershift in the presence of the anti-RXR antibody. Both cell lines were found to express RXRalpha message in equal amounts. Western blot analysis revealed that RXRalpha protein from ras-transformed keratinocytes was indistinguishable from that from immortalized keratinocytes and from control cells. These results suggest a causal relationship between resistance to the growth inhibitory influences of 1,25-dihydroxyvitamin D3 and disruption of the VDR.RXR complex in malignant keratinocytes.     

1,25-dihydroxyvitamin D3 and 9-cis-retinoic acid act synergistically to inhibit the growth of LNCaP prostate cells and cause accumulation of cells in G1

Recent studies have suggested that the active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3, can inhibit the growth and/or induce the differentiation of a variety of cell types and that these characteristics might be useful in the treatment of some cancers. Retinoids also promote the differentiation and inhibit the growth of some cells. That the vitamin D receptor acts as a heterodimer with the retinoid X receptor (RXR) suggests that there may be functional interactions between 1,25-dihydroxyvitamin D3 and retinoids. In this study, we show that the combination of 1,25-dihydroxyvitamin D3 and 9-cis retinoic acid synergistically inhibits the growth of LNCaP prostate cancer cells. That this effect is mediated by RXR rather than retinoic acid receptors was shown using RXR- and retinoic acid receptor-specific ligands. The vitamin D3 analog, EB1089, inhibited growth more effectively than 1,25-dihydroxyvitamin D3 and also acted synergistically with 9-cis-retinoic acid. These treatments caused cells to accumulate in the G1 phase of the cell cycle, suggesting that 1,25-dihydroxyvitamin D3 can regulate one or more factors critical for the G1/S transition.