Trivalent antimonials induce degradation of the PML-RAR oncoprotein and reorganization of the promyelocytic leukemia nuclear bodies in acute promyelocytic leukemia NB4 cells

Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) chromosomal translocation that fuses the genes encoding the promyelocytic leukemia protein (PML) and the retinoic acid receptor (RAR). The resulting PML-RAR protein induces a block in the differentiation of the myeloid progenitor cells, which can be released by retinoic acid (RA) in vitro and in vivo. The RA-induced differentiation of APL blasts is paralleled by the degradation of the fusion protein and the relocation of wild-type PML from aberrant nuclear structures to its normal localization in nuclear bodies. Recently, arsenic trioxide (As2O3) treatment was proposed as an alternative therapy in APL, because it can induce complete remission in both RA-sensitive and -resistant APL patients. Intriguingly, As2O3 was also shown to induce degradation of the PML-RAR chimera and to reorganize PML nuclear bodies. Here we show that trivalent antimonials also have striking effects on RA-sensitive and RA-resistant APL cells. Treatment of the APL-derived NB4 cells and the RA-resistant subclone NB4R4 with antimony trioxide or potassium antimonyl tartrat triggers the degradation of the fusion protein and the concomitant reorganization of the PML nuclear bodies. In addition, as reported for As2O3, the antimonials provoke apoptosis of NB4 and NB4R4 cells. The mechanism of antimony action is likely to be similar to that of As2O3, notably both substances induce the attachment of the ubiquitin-like SUMO-1 molecule to the PML moiety of PML-RAR. From these data, we propose that, in analogy to As2O3, antimonials might have a beneficial therapeutic effect on APL patients, perhaps with less toxicity than arsenic.     

Prevention of autoimmune destruction of syngeneic islet grafts in spontaneously diabetic nonobese diabetic mice by a combination of a vitamin D3 analog and cyclosporine

BACKGROUND: Type 1 diabetes is characterized by the presence of an autoimmune memory, responsible for the destruction of even syngeneic islet grafts. This recurrence of autoimmunity is partly responsible for the need of extensive immunosuppression in pancreas and islet transplantation in type 1 diabetic patients. The aim of the study was to evaluate the capacity of a 20-epi-analog of vitamin D3, KH1060, both alone and in combination with cyclosporine (CsA) to prevent diabetes recurrence in syngeneic islet grafts in nonobese diabetic (NOD) mice. METHODS: Spontaneously diabetic NOD mice grafted with syngeneic islets (n=500) under the kidney capsule were treated with KH1060, CsA, or a combination of both drugs from the day before transplantation until recurrence or 60 days after transplantation. RESULTS: Vehicle-treated mice showed a recurrence of diabetes in 100% of cases (n=17) within 4 weeks. Treatment with high doses of CsA (15 mg/kg/day) or KH1060 (1 microg/kg/2 days) significantly prolonged islet survival (60 days and 50 days, respectively, versus 9.5 days in controls; P<0.001 and P<0.0001). Mice treated with subtherapeutical doses of both drugs combined (KH1060 0.5 microg/kg/2 days + CsA 7.5 mg/kg/day) had significant prolongation of graft survival (48 days; P<0.001) and more importantly, four of five mice that were still normoglycemic 60 days after transplantation showed no recurrence after discontinuation of all treatment. Histology of the grafts of control and combination-treated mice demonstrated that graft infiltration and islet destruction were less severe in grafts of combination-treated mice. Cytokine mRNA analysis in the grafts 6 days after transplantation revealed a clear suppression of interleukin-12 and T helper 1 cytokines and higher levels of interleukin-4 in combination-treated mice. CONCLUSIONS: KH1060, an analog of 1,25(OH)2D3, delays autoimmune disease recurrence after syngeneic islet transplantation in NOD mice, both alone and especially in combination with CsA, possibly restoring tolerance to beta cells in 30% of cases.     

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.     

19-nor vitamin-D analogs: a new class of potent inhibitors of proliferation and inducers of differentiation of human myeloid leukemia cell lines

We have studied the in vitro biological activities and mechanisms of action of 1,25-dihydroxyvitamin D3 (1,25D3) and nine potent 1,25D3 analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common novel structural motiff for almost all the analogs included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentration of analogs producing a 50% clonal inhibition (ED50) ranged between 1 x 10(-9) to 4 x 10(-11) mol/L when using the HL-60 cell line. The most active compound [1, 25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol (Ro 25-9716)] had an ED50 of 4 x 10(-11) mol/L; in contrast, the 1,25D3 produced an ED50 of 10(-9) mol/L with the HL-60 target cells. Ro 25-9716 (10(-9) mol/L, 3 days) was a strong inducer of myeloid differentiation because it caused 92% of the HL-60 cells to express CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT). This compound (10(-8) mol/L, 4 days) also caused HL-60 cells to arrest in the G1 phase of the cell cycle (88% cells in G1 v 48% of the untreated control cells). The p27(kip-1), a cyclin-dependent kinase inhibitor which is important in blocking the cell cycle, was induced more quickly and potently by Ro 25-9716 (10(-7) mol/L, 0 to 5 days) than by 1,25D3, suggesting a possible mechanism by which these analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic leukemia cells cultured with the Ro 25-9716 were also inhibited in their clonal proliferation (ED50, 5 x 10(-11) mol/L) and their expression of CD11b was enhanced (80% positive [10(-9) mol/L, 4 days] v 27% untreated NB4 cells). Moreover, the combination of Ro 25-9716 (10(-9) mol/L) and all-trans retinoic acid (ATRA, 10(-7) mol/L) induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the cells became NBT positive after a similar exposure to the combination of 1,25D3 and ATRA. Surprisingly, Ro 25-9716 also inhibited the clonal growth of poorly differentiated leukemia cell lines (RA-res HL-60 [ED50, 4 x 10(-9) mol/L] and Kasumi-1 [ED50, 5 x 10(-10) mol/L]). For HL-60 cells, Ro 25-9716 markedly decreased the percent of the cells in S phase of the cell cycle and increased the expression of the cyclin-dependent kinase inhibitor, p27(kip-1). In summary, 19-nor vitamin D3 compounds strongly induced differentiation and inhibited clonal proliferation of various myeloid leukemia cell lines, suggesting a therapeutic niche for their use in myeloid leukemia.     

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.     

The PML/RAR alpha oncoprotein is a direct molecular target of retinoic acid in acute promyelocytic leukemia cells

Acute promyelocytic leukemia (APL) is characterized by the translocation, t(15;17) and the expression of a PML/RAR alpha fusion protein that is diagnostic of the disease. There is evidence that PML/RAR alpha protein acts as a dominant negative inhibitor of normal retinoid receptor function and myeloid differentiation. We now show that the PML/RAR alpha fusion product is directly downregulated in response to retinoic acid (tRA) treatment in the human APL cell line, NB4. tRA treatment induces loss of PML/RAR alpha at the protein level but not at the level of mRNA, as determined by Northern blots, by Western blots, and by ligand binding assays and in binding to RA-responsive DNA elements. We present evidence that this regulation is posttranslational. This evidence suggests that tRA induces synthesis of a protein that selectively degrades PML/RAR alpha. We further show that this loss of PML/ RAR-alpha is not limited to the unique APL cell line. NB4, because PML/RAR alpha protein is selectively downregulated by tRA when expressed in the transfected myeloid cell line U937. The loss of PML/RAR alpha may be directly linked to tRA-induced differentiation, because in a retinoid-resistant subclone of NB4, tRA does not decrease PML/RAR alpha protein expression. In NB4 cells, the specific downregulation of the fusion protein decreases the ratio of PML/RAR alpha to wild-type RAR alpha. Because the ratio of expression of PML/RAR alpha to wild-type RAR alpha and PML may be important in maintaining the dominant negative block of myelocytic differentiation, these data suggest a molecular mechanism for restoration by tRA normal myeloid differentiation in APL cells.     

Autocrine regulation of matrix metalloproteinase-9 gene expression and secretion by tumor necrosis factor-alpha (TNF-alpha) in NB4 leukemic cells: specific involvement of TNF receptor type 1

Matrix metalloproteinases have been reported to be involved in tumor cell invasion and metastasis. Dissemination of malignant cells in acute myeloid leukemia (AML) may be mediated by similar mechanisms. Here, we report, that the t(15/17)+ acute promyelocytic leukemia (APL) cell line NB4 constitutively expresses and releases the proenzyme form of matrix metalloproteinase-9 (MMP-9, 92 kDa type IV collagenase/gelatinase, gelatinase B), as well as tissue inhibitor of metalloproteinases-1 (TIMP-1). Both proteins were identified by N-terminal amino acid sequence analysis after purification using gelatin Sepharose affinity chromatography. Whereas 12-O-tetradecanoylphorbol-13 acetate (TPA) increased both MMP-9 and TIMP-1 mRNA levels, tumor necrosis factor-alpha (TNF-alpha) stimulated only MMP-9 gene expression in a dose- and time-dependent manner. Neutralizing monoclonal antibodies (MoABs) to TNF-alpha (anti-TNF-alpha) decreased the constitutive and TPA-dependent expression of MMP-9 but did not influence TIMP-1 expression, either in unstimulated or in TPA-treated NB4 cells. FACS analyses showed that NB4 cells express both TNF receptor 1 (TNF-R1) and TNF-R2 to a similar extent. Blocking MoABs against TNF-R 1 (anti-TNF-R1) decreased the constitutive expression of MMP-9, whereas anti-TNF-R2 had almost no effect. Our results show, that in NB4 cells the expression of MMP-9 but not of TIMP-1 is maintained by autocrine stimulation with TNF-alpha. Thus, leukemic cells may be enabled to leave the bone marrow and infiltrate peripheral tissues by a dysfunction in the regulation of the MMP-9:TIMP-1 equilibrium, possibly triggered through autostimulation by TNF-alpha.     

Chemically induced isomerization and differential uptake modulate retinoic acid disposition in HL-60 cells

The successful introduction of 13-cis-retinoic acid (13-cis-RA) and all-trans-retinoic acid (all-trans-RA) in the chemoprevention and treatment of cancer along with the discovery of different retinoic acid receptors transactivated by different retinoic acid isomers resulted in a number of in vitro studies of the antitumor effects of single retinoic acid isomers. Since the formation of retinoic acid isomers with different receptor affinities might modulate retinoic acid response in vitro, we determined retinoic acid disposition in HL-60 cells and cell culture medium during incubation with 13-cis-, 9-cis-, and all-trans-RA. In medium, retinoic acids underwent a thiol-radical mediated isomerization resulting in a mixture of 13-cis-, 9-cis-, 9,13-di-cis-, and all-trans-RA. Except for the 9, 13-di-cis-RA, all isomers generated in medium were also detected in HL-60 cells. Whereas 9-cis-RA and 13-cis-RA showed similar cellular pharmacokinetics, all-trans-RA reached about fourfold higher concentrations in HL-60 cells compared to 9-cis-RA and 13-cis-RA. Due to its better uptake, all-trans-RA became the main isomer within cells as it was formed in the medium when incubated with 13-cis-RA and 9-cis-RA. Thus, due to the simple chemically induced isomerization and its profound influence on cellular retinoic acid concentrations, studies of the efficacy of single retinoic acid isomers in vitro should be interpreted with caution.     

The role of vitamin D derivatives and retinoids in the differentiation of human leukaemia cells

The capabilities of 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3), and two novel vitamin D analogues, EB1089 and KH1060, to induce the differentiation of two established leukaemia cell lines, U937 and HL-60, were assessed alone or in combination with the retinoid compounds, 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (ATRA). The vitamin D derivatives acted to increase the differentiation of U937 and HL-60 cell cultures in a dose-dependent manner, as determined by nitroblue tetrazolium (NBT) reduction, with EB1089 and KH1060 being more effective than the native hormone. As an additional index of leukaemic cell differentiation, induction of expression of the phenotypic cell surface antigen, CD14, and the beta2-integrins, CD11b and CD18 by the vitamin D and retinoid compounds were monitored using fluorescence activated cell sorting (FACS) analyses. Following 96-hr treatment of U937 and HL-60 cells with 5 x 10(-10) M of the vitamin D derivatives, a striking increase in CD14 antigen expression was apparent, indicating the promotion by these compounds of a monocyte/macrophage lineage of cells. CD11b and CD18 antigen expression were also raised above control levels. In contrast, both retinoid compounds used at the higher concentration of 1 x 10(-8) M were not effective inducers of CD14 antigen expression. However, CD11b and CD18 were both readily increased in U937 and HL-60 cell cultures. Treatment of U937 cell cultures with the vitamin D compounds and the retinoids resulted in cooperative effects on induction of differentiation, with correlation by both NBT reduction and FACS analyses of CD14 antigen expression. The presence of 9-cis RA or ATRA appeared to contribute to the further increase of CD14 in these cells. HL-60 cell cotreatment with these compounds also displayed enhanced cooperative effects in phagocytic function by NBT reduction. However, analysis of CD14 revealed a dramatic diminution in HL-60 cells treated with the combinations of the vitamin D derivatives and the retinoids. Assessment of HL-60 cell morphology treated with these combinations demonstrated the presence of a mixed population of monocytes and granulocytes. CD11b and CD18 antigen expression was also enhanced in both cell lines with cotreatment. The ability of EB1089 and KH1060 to induce leukaemic cell differentiation may provide an additional option for therapeutic use alone or together with other differentiation agents such as 9-cis RA or ATRA.     

地西他滨联合三氧化二砷对NB4细胞凋亡作用的研究

目的 研究去甲基化制剂地西他滨(DAC)单用或联合三氧化二砷(As2O3)对NB4细胞凋亡的作用机制.方法 将不同浓度的DAC、As2O3以及两药联合作用于NB4细胞,不加药为对照组,采用四甲基偶氮唑蓝(MTT)法检测细胞增殖抑制作用,流式细胞术检测细胞凋亡.结果 DAC与As2O3单药对NB4细胞的抑制作用呈浓度时间依赖性(DAC 1 μmol/L作用24、48、72 h的抑制率分别为12.18%、22.72%、35.54%;DAC 2 μmol/L作用24、48、72 h的抑制率分别增高为22.14%、31.18%、45.21%;As2O3 0.5 μmol/L作用24、48、72 h的抑制率分别21.09%、32.43%、44.93%;As2O3 1.0 μmol/L作用24、48、72 h的抑制率分别增高为31.69%、41.12%及54.27%),两药联合抑制作用较单药明显(DAC 1 μmol/L+As2O3 0.5 μmol/L作用24、48、72 h抑制率分别为42.10%、48.75%、60.78%)(P<0.05),各浓度组与对照组比较差异均有统计学意义(P值均<0.05);As2O3 1 μmol/L作用于NB4细胞株48 h可见5.8%的细胞凋亡,联合组增高为17.3%.结论 DAC能显著抑制NB4细胞的增殖并诱导其凋亡,DAC联合As2O3对NB4细胞增殖抑制及诱导凋亡有协同作用.     

Vitamin D derivatives inhibit the mitogenic effects of IGF-I on MCF-7 human breast cancer cells

The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and four novel synthetic analogues (EB1089, KH1060, KH1230 and CB1093) on IGF-I-stimulated growth of MCF-7 human breast cancer cells have been determined. A significant time- and dose-dependent inhibition of IGF-I-stimulated cell growth was seen with EB1089, such that after 7 days of treatment with 10(-8) M EB1089, the mitogenic effect of IGF-I (30 ng/ml) was negated. Comparison with 1,25(OH)2D3 showed the synthetic analogues to be more potent. The anti-oestrogen ICI 182,780 similarly inhibited IGF-I-stimulated growth of these cells and in combination with EB1089 exerted additional inhibitory effects. Retinoids (all-trans-retinoic acid or the isomer 9-cis-retinoic acid) were less effective in limiting MCF-7 cell responsiveness to IGF-I but, in combination with EB1089, a co-operative effect was achieved. Using radioligand-binding techniques, we observed that 1,25(OH)2D3 and EB1089 down-regulated the levels of 125I-IGF-I binding to MCF-7 cell membranes. Scatchard analysis showed that EB1089 decreased maximal binding approximately 2-fold. Vitamin D derivatives were also demonstrated to reduce IGF-I receptor expression in MCF-7 cells by Western analysis. Our findings demonstrate that vitamin D derivatives limit responsiveness of MCF-7 cells to the mitogenic effects of IGF-I, which may be mediated by reduction of IGF-I receptor expression.     

Vitamin K2 and its derivatives induce apoptosis in leukemia cells and enhance the effect of all-trans retinoic acid

Geranylgeraniol, a polyprenylalcohol composing the side chain of vitamin K2 (VK2), was previously reported to be a potent inducer of apoptosis in tumor cell lines (Ohzumi H et al, J Biochem 1995; 117: 11-13). We examined the apoptosis-inducing ability of VK2 (menaquinone 3 (MK3), MK4 and MK5) and its derivatives such as phytonadione (VK1), as well as polyprenylalcohols with side chains of various lengths including farnesol (C15-OH; FO), geranylgeraniol (C20-OH; GGO), and geranylfarnesol (C25-OH; GFO) toward leukemia cells in vitro. MK3, MK4, MK5 and GFO (at 10 microM) showed a potent apoptosis-inducing activity for all freshly isolated leukemia cells tested and for leukemia cell lines such as NB4, an acute promyelocytic leukemia (APL)-derived cell line and MDS92, a cell line derived from a patient with myelodysplastic syndrome, although there were some differences depending on the cells tested. In contrast, VK1 showed no effect on any of the leukemia cells. The combination of MK5 plus all-trans retinoic acid (ATRA) resulted in enhanced induction of apoptosis in both freshly isolated APL cells and NB4 cells as compared to each reagent alone. These data suggest the possibility of using VK2 and its derivatives for the treatment of myelogenous leukemias, including APL.     

1,25-Dihydroxyvitamin D3 and the vitamin D analogue KH1060 induce hyperproliferation in normal mouse epidermis. A BrdUrd/DNA flow cytometric study

1,25-dihydroxyvitamin D3 (calcitriol) affects differentiation and proliferation of epidermal keratinocytes in vitro and in vivo. We have studied the topical effects of calcitriol (0.08-2.0 micrograms/ml) and of a new vitamin D analogue, the epi-20-analogue KH1060 (0.4-2.0 micrograms/ml) on epidermal proliferation in normal hairless mice. Epidermis was examined at intervals from 4 h to 8 days after a single-dose application. The mitotic rate was assessed by the stathmokinetic method and hyperplasia was scored in histological sections. Cell cycle parameters were measured by bivariate bromodeoxyuridine (BrdUrd)/DNA flow cytometry on isolated epidermal basal cells after pulse-labelling with BrdUrd. Both calcitriol and KH1060 induced a dose- and time-dependent increase in the mitotic rate and in hyperplasia, the latter drug being the most effective. Calcitriol and KH1060 induced changes in the cell cycle traverse compatible with the regenerative reaction seen after other hyperplasiogens, but with an additionally increased accumulation of cells in the G2 phase. This is similar to that seen after topical application of retinoic acid to mouse skin. Our results are thus in contrast to the anti-proliferative effects of calcitriol observed in vitro and following treatment of the hyperproliferative disease psoriasis with calcitriol as well as other vitamin D analogues.     

The PML/RARalpha fusion protein inhibits tumor necrosis factor-alpha-induced apoptosis in U937 cells and acute promyelocytic leukemia blasts

We investigated the effect of the acute promyelocytic leukemia (APL) specific PML/RARalpha fusion protein on the sensitivity to TNF-alpha-mediated apoptosis. The U937 leukemia cell line was transduced with PML/RARalpha cDNA. PML/RARalpha expression caused a markedly reduced sensitivity to TNF-alpha, even if apoptosis was triggered by agonistic antibodies to TNF-alpha receptors I and II (TNF-alphaRI, II). PML/RARalpha induced a 10-20-fold decrease of the TNF-alpha-binding capacity via downmodulation of both TNF-alphaRI and TNF-alphaRII: this may mediate at least in part the reduced sensitivity to TNF-alpha. Furthermore, the fusion protein did not modify Fas expression (CD95) or sensitivity to Fas-mediated apoptosis. The pathophysiological significance of these findings is supported by two series of observations. (a) Fresh APL blasts exhibit no TNF-alpha binding and are resistant to TNF-alpha-mediated apoptosis. Conversely, normal myeloblasts-promyelocytes show marked TNF-alphaR expression and are moderately sensitive to TNF-alpha-mediated cytotoxicity. Similarly, blasts from other types of acute myeloid leukemia (AML M1, M2, and M4 FAB types) show an elevated TNF-alpha binding. (b) The NB4 APL cell line, which is PML/RARalpha+, shows low TNF-alphaR expression capacity and is resistant to TNF-alpha-triggered apoptosis; conversely a PML/RARalpha- NB4 subclone (NB4.306) exhibits detectable TNF-alpha-binding capacity and is sensitive to TNF-alpha-mediated cytotoxicity. These studies indicate that the PML/RARalpha fusion protein protects against TNF-alpha-induced apoptosis, at least in part via downmodulation of TNF-alphaRI/II: this phenomenon may play a significant role in APL, which is characterized by prolonged survival of leukemic blasts.     

Combined arsenic and retinoic acid treatment enhances differentiation and apoptosis in arsenic-resistant NB4 cells

In the acute promyelocytic leukemia (APL) cell line NB4, as well as in APL patients' cells, arsenic trioxide (As2O3) leads to incomplete cell maturation, induction of apoptosis, as well as to the degradation of the oncogenic PML/RARalpha fusion protein. We have isolated an arsenic-resistant NB4 subline (NB4-AsR), which fails to undergo apoptosis, but maintains the partial differentiation response to this drug. When grown in the presence of As2O3, NB4-AsR cells degrade PML/RARalpha, slightly differentiate, and become more sensitive to serum deprivation-induced apoptosis. Similarly, in RA-resistant NB4-R1 cells, RA induced a significant PML/RARalpha degradation and yet failed to induce cell maturation. Thus, As2O3- or retinoic acid (RA)-induced PML/RARalpha degradation may be a prerequisite, but is not sufficient for the full differentiative/apoptotic response to these drugs. Strikingly, RA-triggered differentiation and apoptosis were greatly accelerated in As2O3-treated NB4-AsR cells. The synergism between these two agents in this setting could provide an experimental basis for combined or sequential RA/As2O3 therapies.     

All-trans-retinoic acid counteracts endothelial cell procoagulant activity induced by a human promyelocytic leukemia-derived cell line (NB4)

Therapy with all-trans-retinoic acid (ATRA) can rapidly improve the coagulopathy of acute promyelocytic leukemia (APL). This study was designed to evaluate whether the APL cell line NB4 induces the procoagulant activity (PCA) of human endothelial cells (ECs) in vitro, and whether this property is modified after ATRA-induced NB4 maturation. EC monolayers were incubated for 4 hours at 37 degrees C with the conditioned media (CM) of NB4 treated with 1 mumol/L ATRA (ATRA-NB4-CM) or the vehicle (control-NB4-CM). EC lysates were tested for PCA. ATRA-NB4-CM induced significantly more PCA:tissue factor (TF) than control-NB4-CM (P < .01). To identify the cause of TF induction, interleukin (IL)-1 beta antigen levels were measured in CM samples. ATRA-NB4-CM contained significantly more IL-1 beta than control-NB4-CM. EC PCA was significantly inhibited by an anti-IL-1 beta antibody. The addition to the media of 10 mumol/L ATRA counteracted the EC TF expression induced by NB4-CM. These data indicate that ATRA increases the promyelocyte-induced EC TF, partly through increased IL-1 beta production. However, ATRA can protect the endothelium from the procoagulant stimulus of leukemic cells.     

Cardiac troponin I (cTn I) and the postmortem diagnosis of myocardial infarction

KH 1060 is the 20-epi-22-oxa-24a-homo-26,27-dimethyl analogue of the natural hormone, 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3). We have previously shown that after topical application in hairless mice both KH 1060 and 1 alpha,25(OH)2D3 cause epidermal hyperproliferation. MC 1582 differs from KH 1060 by the lack of hydroxyl group in the side chain which is required for receptor binding. We found that MC 1582 strongly stimulates epidermal hyperplasia in hairless mice after topical application in vivo, approaching in potency KH 1060. A similar, although much weaker response was also obtained with 1 alpha-hydroxyvitamin D3 (1 alpha(OH)D3). Since only the vitamin D compounds which possess hydroxyl groups both in the position 1 alpha and in the side chain, bind to the vitamin D receptor, we suggest that a local metabolism of MC 1582 and 1 alpha(OH)D3 takes place in the skin to the active, side-chain-hydroxylated species, probably to KH 1060 and 1 alpha,25(OH)2D3. This study suggests that 1 alpha hydroxylated prodrugs may be of use in the dermatological treatment of the future.