Dexamethasone does not counteract the response of acute promyelocytic leukaemia cells to all-trans retinoic acid

Retinoic acid syndrome is a serious condition that may complicate the treatment of acute promyelocytic leukaemia patients. This syndrome may be treated effectively with high-dose corticosteroid therapy and, as a result, many patients with acute promyelocytic leukaemia receive dexamethasone at some point during treatment. We investigated whether dexamethasone would also antagonize the beneficial effects of retinoic acid. In t(15;17)-positive NB4 cells, dexamethasone did not affect the retinoic acid induced differentiation, normalization of PML-nuclear bodies or the induction of thrombomodulin mRNA. Finally, dexamethasone did not inhibit the anti-proliferative effect of retinoic acid but rather showed anti-proliferative activity itself.     

Thrombocytosis in patients with acute promyelocytic leukaemia during all-trans retinoic acid treatment

In 26 acute promyelocytic leukaemia (APL) patients treated with all- trans retinoic acid (ATRA), 23% had platelet counts between 459 and 800 ×10⁹/l during treatment. These values, observed between days 28 and 45 of ATRA treatment, were transient and asymptomatic. We report two APL cases with platelet counts >1000 × 10⁹/l during ATRA therapy who were treated with recombinant interferon alpha. In both cases ATRA doses were not modified, no complications secondary to thrombocytosis were seen, and they subsequently achieved complete remission. It is suggested that IL-6 may play an important role in the pathogenesis of the thrombocytosis induced by ATRA. To our knowledge, this is the first report of thrombocytosis occurring during ATRA treatment.     

All-trans retinoic acid prevents apoptosis of human marrow CD34+ cells deprived of haematopoietic growth factors

The regulation of apoptosis plays a key role in haematopoiesis. It has been demonstrated that haematopoietic progenitor cells progressively undergo apoptotic cell death in the absence of appropriate growth factors. We studied the effects of pharmacological doses of all-transretinoic acid (ATRA) on the apoptosis of human adult marrow CD34+ progenitor cells cultured for 7 d in a serum-free medium. We quantified CD34+ cells, clonogenic progenitors and 5 week colony-forming cells (CFC) before and after ATRA exposure. Moreover, we defined the apoptotic status of the CD34+ cell fraction by analysis of phosphatidylserine externalization (using annexin V), the relative membrane permeability to 7-aminoactinomycin D (7AAD) and the mitochondrial membrane potential [using 3,3'-dihexyloxacarbocyanine iodide, DiOC6(3)]. In the drastic experimental conditions used, a decrease in viable CD34+ cells, granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E) and 5 week CFC were observed. Exposure to ATRA partially prevented the decrease in viable CD34+, without a concomitant effect on the clonogenic and more immature progenitors. ATRA-treated CD34+ cells displayed changes in apoptotic status compared with control cultures, particularly in lower annexin V-binding. These results were confirmed using 7AAD and DiOC6(3). Our results demonstrate that ATRA exerts a protective effect on CD34+ cells exposed to such apoptotic stress.     

All-trans retinoic acid promotes a differential regulation of adhesion molecules on acute myeloid leukaemia blast cells

Summary. In the present study we investigated the membrance expression of selectin ligands (CD15/Le^x, CDw65/VIM2, CD15s/sLe^x), β2 integrins (CD11a/LFA-1, CD11b/Mac-1) and CD45 phosphatase isoforms (CD45RA, CD45RO on leukaemic cells from 28 patients with acute myeloid malignancies cultured with and without all-trans retinioc acid (ATRA). Within each adhesion system, ATRA was bale to differentially regulate distinct molecules. Furthermore, it was able to exert effects specific for acute promyelocytic leukaemia (APL) blast cells, as well as to induce a series of non-cytotype-restricted phenotypic changes. An impressive feature of ATRA induction was a simultaneous increase in the expression of CD15, CDw65 and CD11b on leukaemic promyelocytes. The sialylated antigen CD15s, however, Showed results independent from the other two carbohydrates (CD15 and CDw65), suggesting a differential enzymatic regulation within the selectin ligands system.
In spite of the well-recognized expression of CD11a throughout all stages of normal myeloid differentiation, APL blast cells were found to virtually lack LFA-1 expression. Moreover, ATRA was unable to promote and up-regulation of this antigen in APL, while inducing a frequent down-modulation in non-APL cases constitutively expressing this antigen. In APL cases ATRA generated an asynchronous phenotype (CD15⁺, CDw65⁺, CD11b⁺, CD11a^-), undetectable on normally maturing myeloid cells, but consistent with the concept that incomplete differentation, in terms of surface molecule expression, can be sufficient to obtain therapeutic results.     

Serum thrombopoietin and erythropoietin levels in patients with acute promyelocytic leukaemia during all-trans retinoic acid treatment

Endogenous serum thrombopoietin (TPO) and various cytokines including erythropoietin (EPO), interleukin (IL)-3, IL-6, IL-11, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF) and stem cell factor (SCF) levels were measured in five patients with acute promyelocytic leukaemia (APL) during all-trans retinoic acid (RA) treatment. During differentiation-inducing therapy, platelet counts slowly increased and reached a peak between days 29 and 46 (median day 35). Serum TPO levels increased parallel to the increasing platelet counts and reached a maximum level during the first 10-20 d of all-trans RA treatment. The circulating TPO levels then decreased in inverse correlation to the platelet counts. These unique changes in serum TPO levels revealed that TPO levels were not regulated by platelet or megakaryocyte mass in patients with APL during differentiation-inducing therapy, and it would appear that TPO levels are directly regulated by all-trans RA during the first 10-20 d of treatment. In addition, the change in circulating EPO levels and reticulocyte counts were similar to that of the TPO levels and platelet counts during all-trans RA treatment, suggesting a close relationship between TPO and EPO signalling.     

Role of P-glycoprotein in all-trans retinoic acid (ATRA) resistance in acute promyelocytic leukaemia cells: analysis of intracellular concentration of ATRA

We analysed the relationship between all-trans retinoic acid (ATRA) resistance and P-glycoprotein (P-gp)-associated multidrug resistance (MDR) in acute promyelocytic leukaemia (APL). There was no difference in the intracellular ATRA accumulation between NB4 cells and an MDR1 cDNA-transduced NB4 subline and between ATRA-resistant NB4 cells (NB4/RA) and an MDR1 cDNA-transduced NB4/RA subline. PSC833, a MDR modifier, did not increase the intracellular accumulation of ATRA or affect the expression of CD11b, the nitroblue tetrazolium (NBT) reduction activity, the proportion of apoptotic cells or the morphology of these four ATRA-treated cell lines. Similar results were obtained in the analysis of APL cells from five patients relapsed after ATRA-induced complete remission.     

All-trans retinoic acid regulates adhesion mechanism and transmigration of the acute promyelocytic leukaemia cell line NB-4 under physiologic flow

The success of all-trans retinoic acid (ATRA) in the therapy of acute promyelocytic leukaemia (APL) has received increased attention. Unfortunately, life-threatening multiorgan failure commonly occurs, i.e. retinoic acid syndrome, and is thought to be the result of organ infiltration by leukaemic cells. We hypothesized that ATRA-induced differentiation of APL cells leads to adhesion receptor alterations responsible for leucocyte extravasation from the blood into tissue. Changes in adhesive properties of the APL cell line NB-4 in response to ATRA were investigated using a parallel plate flow chamber under conditions that recapitulate physiologic flow conditions. Untreated NB-4 cells initially tether and roll on activated human umbilical vein endothelial cell monolayers using a combination of E-selectin, P-selectin and alpha4 integrin. After ATRA treatment, > 80% of initial NB-4 cell attachment to endothelial cells was E-selectin dependent. Stable arrest (firm adherence) of NB-4 cells on activated endothelium was also altered by ATRA treatment. Untreated NB-4 cells used alpha4 integrin to arrest on endothelium, but beta2 integrin dependent arrest was induced by ATRA. With the acquisition of beta2 integrin function, ATRA-treated cells acquired the ability to transmigrate through activated endothelium. Thus, ATRA dramatically altered the adhesion phenotype on NB-4 cells: ATRA induced rolling largely attributable to E-selectin, abrogated alpha4 integrin dependent rolling, and promoted acquisition of beta2 integrin dependent firm adherence and transmigration. These findings represent novel cellular and differentiation effects of ATRA, and, to our knowledge, are the first demonstration that a therapeutic agent differentially regulates alpha4 and beta2 integrin on the same leucocyte.     

Successful use of all-trans retinoic acid in acute promyelocytic leukaemia presenting during the second trimester of pregnancy

Summary. The development of acute leukaemia in pregnancy is a rare event, with an estimated incidence of 0·9–1·2 cases per 100 000 per year and with the majority of cases occurring in the second and third trimesters. We report a case of acute promyelocytic leukaemia (APL) developing during the second trimester of pregnancy which was successfully treated with all-trans retinoic acid (ATRA) resulting in a complete morphological remission.     

Phase I/II study of vaccination with dendritic-like leukaemia cells for the immunotherapy of acute myeloid leukaemia

Twenty-two patients with acute myeloid leukaemia were recruited into a phase I/II clinical trial investigating the vaccination of patients in complete remission (CR) with autologous dendritic-like leukaemia cells (DLLC). At trial entry, leukaemia cells were harvested and tested for their ability to undergo cytokine-induced dendritic cell differentiation. Patients were then treated with intensive chemotherapy. Five patients achieved both CR and had leukaemia cells that successfully underwent differentiation and therefore proceeded to vaccination. Four escalating doses of DLLC were administered weekly by subcutaneous injection. Vaccination was generally well tolerated although one patient developed extensive eczema and an increased antinuclear factor titre possibly indicating induction of autoimmunity. Development of anti-leukaemic T-cell responses was assessed by enzyme-linked immunospot analysis of gamma-interferon secreting T lymphocytes and by human leucocyte antigen tetramer analysis for WT1-specific T cells. Increases in anti-leukaemic T-cell responses were demonstrated in four patients, but only two of the five remained in remission more than 12 months postvaccination. The study has demonstrated that generation of DLLC is feasible in only a subgroup of patients and is currently neither broadly applicable or clinically effective.     

Hypercalcaemia and increased serum interleukin-6 levels induced by all-trans retinoic acid in patients with multiple myeloma

Summary. All-trans retinoic acid (ATRA) inhibits human myeloma cell growth in vitro, presumably through the down-regulation of interleukin 6 receptors (IL-6R). Based on these and other studies, we initiated a phase II clinical trial using ATRA in patients with advanced refractory multiple myeloma (MM). We report that three out of six treated patients developed severe hypercalcaemia following administration of ATRA, which was accompanied by a significant rise in serum IL-6 levels. Normal calcium levels were restored after the discontinuation of the drug and the administration of standard anti-hypercalcaemic care. We suspect that down-regulation of IL-6R resulted in increased serum IL-6 levels, leading to advanced bone resorption and hypercalcaemia. We conclude that the use of ATRA in patients with advanced MM is not warranted.     

All-trans retinoic acid-induced downregulation of annexin II expression in myeloid leukaemia cell lines is not confined to acute promyelocytic leukaemia

Most acute promyelocytic leukaemia (APL) patients suffer from disordered haemostasis. APL can be treated successfully in most instances by all-trans retinoic acid (ATRA) therapy, which induces endpoint maturation of the leukaemic promyelocytes with the characteristic t(15;17). Annexin II (AnII), a profibrinolytic protein, has been implicated in the bleeding manifestation seen in APL. Our group has shown previously that high levels of AnII are expressed on other acute myeloid leukaemia subtypes that are sometimes associated with disordered haemostasis, albeit less frequently than APL. This study examined the effects of ATRA on AnII expression and cell differentiation, on myeloid leukaemia cell lines to determine whether a regulatory influence on AnII may contribute to the return of haemostatic stability in APL following treatment. The results confirmed that AnII expression in the APL cell line (NB4) was significantly downregulated in response to ATRA (P < 0.01), with associated morphological and immunophenotypical evidence of myeloid differentiation. ATRA also downregulated AnII expression on other myeloid cell lines, albeit to a lesser extent than observed on NB4 cells. The results provide evidence that ATRA may resolve the hyperfibrinolysis in APL by downregulation of AnII expression.     

Respiratory failure during induction chemotherapy for acute myelomonocytic leukaemia (FAB M4Eo) with ara-C and all-trans retinoic acid

We report two cases of acute myeloid leukaemia FAB classification M4Eo with high white cell counts at presentation, who developed acute respiratory failure with pulmonary infiltrates on chest radiograph soon after commencing conventional cytotoxic chemotherapy plus all-trans retinoic acid (ATRA). We suggest that in patients with M4Eo ATRA should be used with caution, perhaps delaying its commencement until the white cell count is < 10 x 109/l.     

Clinical and laboratory evaluation of all-trans retinoic acid modulation of chemotherapy in patients with acute myelogenous leukaemia

All-trans retinoic acid (ATRA) is synergistic with chemotherapy in leukaemia cell lines. We treated 53 patients with newly diagnosed acute myelogenous leukaemia (AML) with high-dose cytarabine-based chemotherapy followed by ATRA. Peripheral blood and bone marrow samples were obtained to study the effect of in vitro exposure to ATRA and to measure apoptosis and bcl-2. The response rate was 72% for patients under age 60 years and 46% for patients aged 60 years or above. There was no difference in the percentage of responding patients, time to recurrence or overall survival for patients receiving chemotherapy with ATRA vs. historical controls receiving chemotherapy without ATRA. After in vitro exposure of day 3 bone marrow samples to ATRA, there was an increase in apoptotic cells in 25% of patient samples compared with samples not exposed to ATRA. Later date of peak apoptosis in peripheral blood and higher percentage of apoptotic cells in bone marrow on day 3 of treatment were associated with lack of clinical response to treatment. Increased bcl-2 in patient samples was associated with shorter time to recurrence and poor cytogenetic risk. The addition of ATRA to chemotherapy did not improve patient outcome. However, evidence of in vitro response to ATRA in 25% of patients suggests that retinoid pathways should be studied further in patients with AML.     

Histone deacetylase inhibitor but not arsenic trioxide differentiates acute promyelocytic leukaemia cells with t(11;17) in combination with all-trans retinoic acid

Acute promyelocytic leukaemia (APL) with t(11;17)/PLZF-RARalpha responds poorly to all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3), in contrast to APL with t(15;17)/PML-RARalpha. Molecular studies have shown that histone deacetylase (HDAC) recruited by PLZF-RARalpha is associated with the ATRA resistance. Here, we analysed in vitro the differentiation of APL cells with t(11;17) using ATRA, As203, granulocyte colony-stimulating factor (G-CSF), HDAC inhibitor trichostatin A (TSA), or combinations of these. Although 1 microM ATRA, which stimulated the differentiation of APL cells with t(15;17), was insufficient to induce differentiation, 3 microM ATRA induced terminal differentiation into granulocytes. As203 alone or in combination with ATRA induced neither differentiation nor apoptosis. However, the combination of TSA and 1 microM ATRA had a potent differentiating effect, although TSA alone had little effect. The combination of 1 microM ATRA and G-CSF did not induce differentiation. These results indicate that APL cells with t(11;17) need a higher concentration of ATRA than those with t(15;17) to differentiate and suggest that HDAC inhibitor is a promising differentiation enhancer in APL with t(11;17).     

Early haemorrhagic morbidity and mortality during remission induction with or without all-trans retinoic acid in acute promyelocytic leukaemia

A total of 622 consecutive patients with acute promyelocytic leukaemia (APL) treated within the Gruppo Italiano per le Malattie Ematologiche dell'Adulto (GIMEMA) group during 1989-97 have been reviewed to assess the clinical effectiveness of all-trans retinoic acid (ATRA) on the incidence of early haemorrhagic deaths and on APL-associated coagulopathy. Of them, 499 were treated with idarubicin plus ATRA (study A) and 123 with Idarubicin alone (study B). In both studies, similar guidelines for supportive treatment were used. Haemorrhagic symptoms were evaluated according to a reproducible score system. Deaths occurring within 10 d of starting treatment were 19 (3.8%) in study A and nine (7.3%) in study B (P = 0.09), with 15 (3%) and five (4.1%) (P not significant) due to haemorrhage. Overall, induction mortality was 7.6% and 16.2% respectively (P < 0.003). In study A, days with platelet counts 30 x 109/l (P < 0.001) in both studies, and by a haemorrhagic score of 3 in study A (P < 0.001). Although the reduction of early fatal haemorrhages was not significant, a substantial clinical improvement was evident in terms of reduction of the severity of bleeding symptoms, blood product consumption and overall induction mortality when ATRA was combined with idarubicin.     

Conversion of Th2 memory cells into Foxp3+ regulatory T cells suppressing Th2-mediated allergic asthma

Genetic and epigenetic programming of T helper (Th) cell subsets during their polarization from naive Th cells establishes long-lived memory Th cells that stably maintain their lineage signatures. However, whether memory Th cells can be redifferentiated into another Th lineage is unclear. In this study, we show that Ag-specific memory Th cells were redifferentiated into Foxp3⁺ T cells by TGF-β when stimulated in the presence of all-trans retinoic acid and rapamycin. The “converted” Foxp3⁺ T cells that were derived from Th2 memory cells down-regulated GATA-3 and IRF4 and produced little IL-4, IL-5, and IL-13. Instead, the converted Foxp3⁺ T cells suppressed the proliferation and cytokine production of Th2 memory cells. More importantly, the converted Foxp3⁺ T cells efficiently accumulated in the airways and significantly suppressed Th2 memory cell-mediated airway hyperreactivity, eosinophilia, and allergen-specific IgE production. Our findings reveal the plasticity of Th2 memory cells and provide a strategy for adoptive immunotherapy for the treatment of allergic diseases.     

全反式维甲酸对卵巢癌SKOV3细胞增殖、分化的影响

目的 探讨全反式维甲酸（ATRA）对卵巢癌SKOV3细胞增殖、分化的影响.方法 以1×10^-6 mol/L的ATRA处理人卵巢癌SKOV3细胞为实验组,细胞不加ATRA处理为对照组.应用MTT法测定细胞增殖抑制率,流式细胞仪测定细胞周期分布,免疫组化法检测鼠抗人分化相关基因1（NDRG1）表达,显微镜观察细胞形态变化.结果 与对照组比较,实验组细胞增殖抑制率、NDRG1表达升高（P均＜0.05）;随着作用时间延长,实验组G/G0期细胞增多、S期细胞减少,显微镜下可见细胞生长差,增殖缓慢,分裂像少见.结论 ATRA有较强的抑制卵巢癌SKOV3细胞增殖和诱导其分化作用.