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.     

Differential regulation of GPI-linked molecules on leukaemic promyelocytes treated in vitro with all-trans retinoic acid

It has been demonstrated that certain cell-surface proteins are anchored to the cell membrane by a unique structure known as the glycosylphosphatidylinositol (GPI) anchor whose absence has been reported on blood cells from patients with paroxysmal nocturnal haemoglobinuria. We have investigated the expression of CD16/FcτR-III and CD66b GPI-linked molecules at the surface of blast cells from five acute promyelocytic leukaemia (APL) patients before and after in vitro stimulation with all-trans retinoic acid (ATRA). We observed that whereas CD66b antigen exhibited a strong ATRA-driven up-regulation in all cases studied, CD16 expression was unaffected by the treatment with the drug.     

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.     

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.     

Rapid improvement of coagulopathy by all-trans retinoic acid in acute promyelocytic leukemia

Treatment of acute promyelocytic leukemia (APL) patients with all-trans retinoic acid (ATRA) was associated with rapid improvement in hemostatic markers. We made serial analyses of various hemostatic parameters in seven newly diagnosed APL patients. In all patients at diagnosis, plasma fibrinogen/fibrin degradation product (fragment-E), cross-linked fibrin degradation product (D-dimer fragment), thrombin-antithrombin III complex and plasmin-α2-plasmin inhibitor complex were elevated, indicating the presence of disseminated intravascular coagulation (DIC). Antithrombin III (A TIII) levels were normal in all patients except for the patient with congenital ATIII deficiency. In four patients subsequently treated with ATRA without anticoagulant therapy, these hemostatic markers returned to near-normal levels by day 7 of treatment, indicating that DIC was essentially resolved. By contrast, in three patients who received conventional chemotherapy with a continuous low-dose heparin, improvement of coagulopathy was slower than in patients treated with ATRA. These results suggest that ATRA therapy exerts the rapid improvement in abnormal hemostatic markers in APL patients without any anticoagulant therapies, by inducing differentiation of leukemic cells and, in turns no massive release of procoagulant or fibrinolytic substances from these cells. © 1994 Wiley-Liss, Inc.     

Changes in erythroid progenitor cell and accessory cell compartments in patients with myelodysplastic syndromes during treatment with all-trans retinoic acid and haemopoietic growth factors

Differentiation induction therapy is used in myelodysplastic syndromes (MDS) to improve maturation defects and to restore impaired function of malignant cells. To this end, 18 patients with MDS received either a combination therapy consisting in study 1 of all- trans retinoic acid (ATRA) and granulocyte-colony stimulating factor (G-CSF), or in study 2 of a combination with ATRA, G-CSF, erythropoletin (Epo) and tocopherol. The ANC increased in 19/20 patients in both studies, whereas an increase in haemoglobin concentration, platelet counts or reduction of transfusion requirement was seen in only 8/20 patients, correlating strongly with good BFU-E growth ( P < 0.001). To assess the role of accessory cells in the modulation of the haemopoietic response to treatment, we analysed the capacity of peripheral blood monocytes to secrete cytokines (IL-1β, IL-6, IL-8, TNFα). Secretion of all cytokines was significantly reduced before therapy when compared with healthy controls, but increased during therapy, reaching normal levels for IL-8. These data indicate that a combination therapy with ATRA and cytokines improves impaired cytokine secretion from monocytes and induces a multilineage clinical response in a subgroup of MDS patients characterized by an almost intact erythroid compartment. In contrast, induction of TNFα might be responsible for treatment failure.     

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.     

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.     

全反式维甲酸对大鼠肾脏表面活性蛋白D表达的影响

目的探讨全反式维甲酸（atRA）对肾盂肾炎大鼠肾脏表面活性蛋白D（SP-D）表达的影响,以及肾脏炎症程度与SP-D表达之间的关系。方法将24只SPF级雌性Wistar大鼠随机分为4组：正常对照组、假手术组、肾盂肾炎模型组、肾盂肾炎＋atRA治疗组。采用致肾盂肾炎大肠杆菌肾筋膜下注射法制造肾盂肾炎模型。HE染色法观察肾组织病理改变,免疫组化法观察SP-D表达部位及强度。结果与对照组相比,模型组炎症评分和SP-D表达强度均显著增加,治疗组炎症评分和SP-D表达强度也显著增加,但低于模型组（P均〈0.05）,假手术组与对照组相比无统计学差异（P〉0.05）。肾脏炎症程度与SP-D表达强度呈正相关（r=0.842,P〈0.05）。结论SP-D可能在肾脏的先天免疫与炎症反应中有重要调节作用;atRA可能通过下调SP-D表达,减轻肾脏炎症损伤程度。     

全反式维甲酸对人喉癌细胞株Hep-2体外增殖及Survivin表达的影响

目的 进一步探讨全反式维甲酸（ATRA）对人喉癌的治疗机制。方法取对数生长期人喉癌细胞株Hep-2,以1×10^5／ml细胞密度接种于96孔培养板,置于CO2培养箱中24h后随机分为观察组和对照组,观察组分别加入终浓度为10^-7、10^-6、10^-5mol／L的ATRA,对照组为空白对照。观察两组细胞增殖抑制率（IR）、细胞凋亡率、Survivin表达水平。结果观察组Hep-2细胞IR及细胞凋亡率均显著高于对照组,Survivin表达显著低于对照组,且呈时间和剂量依赖性。结论ATRA对人喉癌细胞株Hep-2生长有抑制作用,同时可诱导其凋亡,机制可能与Survivin基因表达下调有关。     

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.     

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.     

Prognostic value of FLT3 mutations in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline monochemotherapy

Background

Fms-like tyrosine kinase-3 (FLT3) gene mutations are frequent in acute promyelocytic leukemia but their prognostic value is not well established.

Design and Methods

We evaluated FLT3-internal tandem duplication and FLT3-D835 mutations in patients treated with all-trans retinoic acid and anthracycline-based chemotherapy enrolled in two subsequent trials of the Programa de Estudio y Tratamiento de las Hemopatías Malignas (PETHEMA) and Hemato-Oncologie voor Volwassenen Nederland (HOVON) groups between 1996 and 2005.

Results

FLT3-internal tandem duplication and FLT3-D835 mutation status was available for 306 (41%) and 213 (29%) patients, respectively. Sixty-eight (22%) and 20 (9%) patients had internal tandem duplication and D835 mutations, respectively. Internal tandem duplication was correlated with higher white blood cell and blast counts, lactate dehydrogenase, relapse-risk score, fever, hemorrhage, coagulopathy, BCR3 isoform, M3 variant subtype, and expression of CD2, CD34, human leukocyte antigen-DR, and CD11b surface antigens. The FLT3-D835 mutation was not significantly associated with any clinical or biological characteristic. Univariate analysis showed higher relapse and lower survival rates in patients with a FLT3-internal tandem duplication, while no impact was observed in relation to FLT3-D835. The prognostic value of the FLT3-internal tandem duplication was not retained in the multivariate analysis.

Conclusions

FLT3-internal tandem duplication mutations are associated with several hematologic features in acute promyelocytic leukemia, in particular with high white blood cell counts, but we were unable to demonstrate an independent prognostic value in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline-based regimens.     

Marked basophilia in acute promyelocytic leukaemia treated with all-trans retinoic acid: molecular analysis of the cell origin of the basophils

Summary. We report a patient with acute promyelocytic leukaemia who developed marked basophilia during all-trans retinoic acid treatment. We studied genomic DNA and RNA extracted from the patient's peripheral leucocytes in order to determine the origin of the basophils. The RARα rearranged band in the Southern blot analysis and a chimaeric product of PML-RARα by polymerase chain reaction were strongly visible before ATRA treatment, but at the time of maximal basophilia both of them were markedly diminished. These findings suggest that the basophils which appeared during the ATRA treatment are reactive in nature rather than a leukaemic clone.     

全反式维甲酸对转化生长因子诱导的肾小球系膜细胞环氧化酶2表达的影响

目的:研究仝反式维甲酸(all-trans retinoic acid,ATRA)对转化生长因子β(transforming growth factor-β,TGF-β)刺激系膜细胞环氧化酶2(COX-2)及Smad3,Smad7蛋白表达的影响,探讨ATRA在TGF-β/Smad信号通路中对系膜细胞表达COX-2的影响作用. 方法:取培养第4代的大鼠肾小球系膜细胞分组:(1)对照组;(2)TGF-β刺激组(5、10 ng/ml);(3)TGF-β+NS398[COX-2抑制剂组(10 μmol/L NS398+10 ng/m 1TGF-β)];(4)TGF-β+Staurosporine(Smad抑制剂)组(5 nmol/ml Staurosporine+10 ng/ml TGF-β);(5)药物组:ATRA(10~(-3)、10~(-6)、10~(-9)moL/L)组,在不同的作用时间(4h,12h,24h),Western Blot印迹法测定各组COX-2、Smad3、Smad7蛋白的表达变化. 结果:(1)在外源性TGF-β刺激下,COX-2、Smad3、Smad7蛋白的表达明显增加,各组与对照组比较,差异有统计学意义(P<0.05).(2)加入COX-2抑制剂NS-398和Smad抑制剂Staurosporine后,COX-2、Smad3、Smad7表达均减少,与TGF-β组比较,差异有统计学意义(P<0.05).(3)ATRA各浓度组和TGF-β组比较COX-2、Smad3、Smad 7蛋白的表达明显减少(P<0.05),呈时间和浓度依赖性.(4)COX-2蛋白的表达与Smad3、Smad7蛋白的表达均呈显著正相关(r=0.978,r=0.942,P<0.05). 结论:ATRA可能通过TGF-β/Smad信号通路抑制系膜细胞COX-2的表达,显示其具有良好的抗纤维化和抗细胞增殖作用.     

Simultaneous induction of matrix metalloproteinase-9 and interleukin 8 by all-trans retinoic acid in human PL-21 and NB4 myeloid leukaemia cells

All-trans retinoic acid (ATRA) has been shown to induce differentiation of human acute promyelocytic leukaemia (APL) cells and eventual elimination of the malignant clone. Matrix metalloproteinase-9 (MMP-9) is produced by neutrophils and its expression appears to be linked with myeloid cell differentiation. We investigated effects of ATRA on MMP expression in two human myeloid leukaemia cell lines, PL-21 and NB4. Both cells could differentiate into neutrophils after exposure to ATRA. Both the activity and antigen levels of MMP-9 were much higher in NB4 cells than in PL-21 cells. Stimulation with ATRA significantly increased MMP-9 levels approximately three- to fivefold in both PL-21 and NB4-conditioned media. MMP-9 mRNA levels increased in ATRA-treated cells and was almost in parallel with the increase in MMP-9 activity, suggesting that ATRA induced MMP-9 by activating its gene expression. ATRA can induce interleukin 8 (IL-8) in APL cells. IL-8, chemokine for neutrophils and a potent inducer of MMP-9, was also induced by ATRA in PL-21 cells. However, recombinant IL-8 did not induce MMP-9 expression. In addition, a neutralizing antibody against IL-8 did not inhibit ATRA-induced MMP-9 expression in either cell type. These observations suggest that ATRA can induce both MMP-9 and IL-8, but IL-8 is not involved in ATRA-induced MMP-9 expression. As MMP-9 can truncate and activate IL-8, simultaneous induction of MMP-9 and IL-8 by ATRA could activate leucocytes excessively, causing the hyper-inflammatory events in retinoic acid syndrome.     

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).     

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.     

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.