维甲酸诱导的人食管癌EC—109细胞分化相关基因的克隆

目的：克隆维甲酸诱导的人食管癌ＥＣ－１０９细胞分化相关基因。方法：在全反式维甲酸诱导人食管癌ＥＣ－１０９细胞系分化的基础上，采用ｍＲＮＡ差异显示技术（ｍＲＮＡｄｉｆｆｅｒｅｎｔｉａｌｄｉｓｐｌａｙ，ＤＤ）对ＥＣ－１０９细胞全反式维甲酸诱导前后的基因表达差异情况进行了分析。结果：全反式维甲酸诱导前后的细胞之间存在明显的基因表达差异，经克隆筛选获得了一些经全反式维甲酸诱导激活或抑制的差异表达基因片段。对其中一个片段（ＲＡａｌ２）的序列分析及同源性比较结果表明其与人的ａｌｐｈａ－ｃａｔｅｎｉｎ基因（Ｄ１３８６６，ＨＵＭＡＣＡ，２４３９ｂｐ）１００％同源。结论：全反式维甲酸具有调控分化相关基因表达的重要作用，在全反式维甲酸诱导前后的细胞之间存在明显的基因表达差异     

Additive Inhibitory Effect of Experimentally Induced Hepatic Cirrhosis by Agonists of Peroxisome Proliferator Activator Receptor γ and Retinoic Acid Receptor

Peroxisome proliferator activator receptor (PPAR) ligands prevent liver fibrosis, while the role of all-trans retinoic acid (ATRA) and its metabolite 9-cis retinoic acid (9-cis RA) is less clear. We have investigated the ability of the combination of PPARγ ligand rosiglitazone (RSG) and of ATRA to prevent liver fibrosis. In vivo treatment with RSG or ATRA reduced fibrotic nodules, spleen weight, and hydroxyproline levels in rat model of thioacetamide-induced liver fibrosis. The combination of ATRA + RSG caused the strongest inhibition, accompanied by decreased expression of collagen I, α-smooth muscle actin, TGFβ1, and TNFα. In vitro studies showed that PPARγ ligand 15-deoxy-Δ12,14-prostaglandinJ(2)[PJ(2)] and RXR ligand 9-cis RA or PJ(2) and ATRA inhibited proliferation of hepatic stellate cells HSC-T6. 9-cis RA inhibited c-jun levels and also inhibited expression of its receptor RXRα in HSC-T6 cells. The combination of PPAR-γ and RAR agonists demonstrated an additive effect in the inhibition of TAA-induced hepatic fibrosis, due to inhibition of HSC proliferation and reduction of profibrotic TGFβ1 and proinflammatory TNFα.     

All-trans and 9-cis retinoic acid alter rat hepatic stellate cell phenotype differentially

BACKGROUND: Hepatic stellate cells exert specific functions in the liver: storage of large amounts of retinyl esters, synthesis and breakdown of hepatic extracellular matrix, secretion of a variety of cytokines, and control of the diameter of the sinusoids. AIMS: To examine the influence of all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9RA) on extracellular matrix production and proliferation of activated hepatic stellate cells. METHODS: Cells were isolated using collagenase/pronase, purified by centrifugation in nycodenz, and cultured for two weeks. At this time point the cells exhibited the activated phenotype. Cells were exposed to various concentrations of ATRA and 9RA. The expression of procollagens I, III, and IV, of fibronectin and of laminin were analysed by immunoprecipitation and northern hybridisation. RESULTS: ATRA exerted a significant inhibitory effect on the synthesis of procollagens type I, III, and IV, fibronectin, and laminin, but did not influence stellate cell proliferation, whereas 9RA showed a clear but late effect on proliferation. 9RA increased procollagen I mRNA 1.9-fold, but did not affect the expression of other matrix proteins. CONCLUSION: Results showed that ATRA and 9RA exert different, often contrary effects on activated stellate cells. These observations may explain prior divergent results obtained following retinoid administration to cultured stellate cells or in animals subjected to fibrogenic stimuli.     

The aberrant fusion proteins PML-RAR alpha and PLZF-RAR alpha contribute to the overexpression of cyclin A1 in acute promyelocytic leukemia

Cyclin A1 is a newly discovered cyclin that is overexpressed in certain myeloid leukemias. Previously, the authors found that the frequency of cyclin A1 overexpression is especially high in acute promyelocytic leukemia (APL). In this study, the authors investigated the mechanism of cyclin A1 overexpression in APL cells and showed that the APL-associated aberrant fusion proteins (PML-retinoic acid receptor alpha [PML-RAR alpha] or PLZF-RAR alpha) caused the increased levels of cyclin A1 in these cells. The ectopic expression of either PML-RAR alpha or PLZF-RAR alpha in U937 cells, a non-APL myeloid cell line, led to a dramatic increase of cyclin A1 messenger RNA and protein. This elevation of cyclin A1 was reversed by treatment with all-trans retinoic acid (ATRA) in cells expressing PML-RAR alpha but not PLZF-RAR alpha. ATRA also greatly reduced the high levels of cyclin A1 in the APL cell lines NB4 and UF-1. No effect of ATRA on cyclin A1 levels was found in the ATRA-resistant NB4-R2 cells. Further studies using ligands selective for various retinoic acid receptors suggested that cyclin A1 expression is negatively regulated by activated RAR alpha. Reporter assays showed that PML-RAR alpha led to activation of the cyclin A1 promoter. Addition of ATRA inhibited PML-RAR alpha-induced cyclin A1 promoter activity. Taken together, our data suggest that PML-RAR alpha and PLZF-RAR alpha cause the high-level expression of cyclin A1 seen in acute promyelocytic leukemia. (Blood. 2000;96:3894-3899)     

Differentiation-independent retinoid induction of folate receptor type beta, a potential tumor target in myeloid leukemia

Folate receptor (FR) type beta is expressed in the myelomonocytic lineage, predominantly during neutrophil maturation and in myeloid leukemias. FR-beta expression was elevated up to 20-fold by all-trans retinoic acid (ATRA) in KG-1 myeloid leukemia cells in a dose-dependent and reversible manner in the absence of terminal differentiation or cell growth inhibition. ATRA also increased FR-beta expression in vitro in myeloid leukemia cells from patient marrow. FR-beta was not up-regulated in KG-1 cells treated with phorbol ester, dexamethasone, 1,25-dihydroxy vitamin D(3), or transforming growth factor beta. ATRA did not induce FR-beta expression in receptor negative cells of diverse origin. The ATRA-induced increase in FR-beta expression in KG-1 cells occurred at the level of messenger RNA synthesis, and in 293 cells containing a stably integrated FR-beta promoter-luciferase reporter construct, ATRA induced expression of the reporter. From experiments using retinoid agonists and antagonists and from cotransfection studies using the FR-beta promoter and expression plasmids for the nuclear receptors retinoic acid receptor (RAR)alpha, RARbeta, or RARgamma, it appears that the retinoid effect on FR-beta expression could be mediated by ligand binding to RARs alpha, beta, or gamma, but not to retinoid X receptors. Furthermore, there was apparent cross-talk between RARalpha and RARgamma selective agonists or antagonists, suggesting a common downstream target for RAR isoforms in inducing FR-beta expression. Thus, blocks in the RARalpha-specific pathway of retinoid-induced differentiation may be bypassed during retinoid induction of FR-beta expression. The results suggest that to facilitate FR-targeted therapies, retinoids may be used to modulate FR-beta expression in myeloid leukemia cells refractory to retinoid differentiation therapy.     

复方青黛片联合全反式维甲酸治疗急性早幼粒细胞白血病的近期临床疗效观察

目的:观察复方青黛片联合全反式维甲酸治疗急性早幼粒细胞白血病(APL)的临床疗效、作用特点及不良反应。方法:将APL患者随机分22例,21例及18例3组,分别给予全反式维甲酸(ATRA),复方青黛片及ATRA联合复方青黛片治疗,观察APL的缓解率,无病生存期及药物不良反应。结果:联合治疗组可减少达缓解所需时间,较快速恢复凝血功能。结论:复方青黛片联合全反式维甲酸治疗APL是一种更为有效及合理的方法,是切实可行的,值得推广。     

Acute myeloid leukemia with myelodysplasia-related changes mimicking acute promyelocytic leukemia

We describe a patient with acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) that clinically resembled acute promyelocytic leukemia (APL). The karyotype of his leukemic cells was 46, XY, del (3) (q?) and did not include a chromosomal translocation involving the retinoic acid receptor-α gene. However, retinoic acid syndrome developed, and partial remission was achieved after treatment with all-trans retinoic acid (ATRA) followed by chemotherapy. Our case might provide new insights into the mechanism of the growth inhibitory effect of ATRA on APL-like cells.     

Remodeling of white adipose tissue after retinoic acid administration in mice

A reduced brown adipose phenotype in white adipose tissue (WAT) may contribute to obesity and type 2 diabetes in humans. Retinoic acid, the carboxylic form of vitamin A, triggers in rodents a reduction of body weight and adiposity and an increased expression of uncoupling proteins in brown adipose tissue and skeletal muscle. In this study, we investigated possible remodeling effects of all-trans retinoic acid (ATRA) in WAT depots. Changes in the expression of genes related to thermogenesis and fatty acid oxidation and levels of phosphorylated retinoblastoma protein were analyzed in WAT depots of adult NMRI male mice acutely injected with ATRA or vehicle, together with biometric and blood parameters. Body fat loss after ATRA treatment was unaccompanied by any increase in circulating nonesterified fatty acids or ketone bodies and accompanied by increased rectal temperature. The treatment triggered an up-regulation of the mRNA levels of uncoupling proteins 1 and 2, peroxisome proliferator-activated receptor gamma coactivator-1alpha, peroxisome proliferator-activated receptor alpha, muscle- and liver-type carnitine palmitoyltransferase 1, and subunit II of cytochrome oxidase in different WAT depots. Levels of phosphorylated retinoblastoma protein in WAT depots were increased after ATRA treatment. Adipocyte size was reduced, and the number of multilocular adipocytes was increased in inguinal WAT of ATRA-treated mice. The results indicate that ATRA favors the acquisition of brown adipose tissue-like properties in WAT. Understanding the mechanisms and effectors involved in the remodeling of WAT can contribute to new avenues of prevention and treatment of obesity and type 2 diabetes.     

Maintenance of retinoic acid receptor alpha pools by granulocyte colony-stimulating factor and lithium chloride in all-trans retinoic acid-treated WEHI-3B leukemia cells: relevance to the synergistic induction of terminal differentiation

Previous studies have demonstrated that combinations of all-trans retinoic acid (ATRA) with either granulocyte-colony stimulating factor (G-CSF) or lithium chloride (LiCl) produced synergistic terminal differentiation of WEHI-3B myelomonocytic leukemia (D(+)) cells. It was found that steady-state retinoic acid receptor alpha (RARalpha) protein levels were markedly reduced in these cells after exposure to ATRA. Because the presence of receptors for a hormone ligand is required for its action, differentiation therapy with ATRA may be self-limiting. The combination of G-CSF with ATRA significantly attenuated the loss of RARalpha protein, and synergistic terminal differentiation occurred. LiCl was more effective than G-CSF in preserving RARalpha pools and synergized with ATRA more strongly than G-CSF. These findings suggested that the prevention of RARalpha protein loss by G-CSF or LiCl in ATRA-treated cells functioned to extend the differentiation response to the retinoid and was responsible, at least in part, for the observed synergism. D(+) cells transfected with an expression plasmid containing RARalpha cDNA had a 6- to 8-fold increase in steady-state RARalpha mRNA compared with vector-transfected cells and showed a 2- to 3-fold increase in RARalpha protein. ATRA caused a reduction, but not a complete loss, of RARalpha protein in these transfectants, which were considerably more responsive than parental D(+) cells to ATRA as a single agent, supporting the concept that the protection of RARalpha pools results in a heightened differentiation response to ATRA.     

抑癌基因p16对维A酸诱导肺癌细胞分化作用的影响

目的探讨抑癌基因p16在维A酸(RA)对肺癌细胞的增殖抑制和分化调控过程中的作用.方法运用基因转染技术将抑癌基因p16转入人肺癌细胞A549中,进而以浓度为5×10-6mol/L的全反式维A酸(ATRA)处理转染和未转染p16基因的肺癌A549细胞1～4d,观察A549细胞生长,流式细胞仪进行细胞周期分析,应用免疫组化分析肺组织分化标志物粘蛋白(MUC1)的变化,同时Westernblot观察ATRA对P16蛋白表达的影响.结果转染p16抑癌基因的A549肺癌细胞经ARTA处理后,细胞增殖能力明显下降,更多的细胞被阻滞于G1/G0期,MUC1的表达下调,Westernblot表明经ATRA处理后P16蛋白表达增加.结论抑癌基因p16能协同RA抑制肺癌A549细胞的恶性增殖及诱导其分化.     

All-trans retinoic acid in acute promyelocytic leukaemia

The vitamin A derivative, all-trans retinoic acid (ATRA), induces differentiation of leukaemic promyelocytes in patients with acute promyelocytic leukaemia (APL). As a result, the majority of patients achieve complete remission either with ATRA alone or with combined ATRA and chemotherapy. The most important complication is the retinoic acid syndrome, which is usually successfully treated with the early administration of dexamethasone. Prospective randomized trials have shown that ATRA is better than conventional chemotherapy in newly diagnosed patients, that ATRA combined with chemotherapy confers an advantage with respect to relapse rate, compared to ATRA alone for induction followed by chemotherapy for consolidation, and that maintenance therapy with ATRA or ATRA plus low-dose chemotherapy is beneficial. The presence of adverse prognostic factors, including older age, presenting white blood cell count and platelet count, expression of CD56 and presence of mutations in the FLT3 gene, identify patients at risk for relapse for whom new strategies are needed.     

Peroxisome Proliferator-Activated Receptor γ and Retinoic Acid Receptor Synergistically Up-Regulate the Tumor Suppressor PTEN in Human Promyeloid Leukemia Cells

Peroxisome proliferator-activated receptor gamma (PPARgamma) and retinoic acid receptors (RARs) have been a focus in chemotherapy for human cancers. The tumor suppressor PTEN plays a pivotal role in the growth of human cancer cells. We investigated whether costimulation of PPARgamma and RAR could synergistically up-regulate PTEN in human leukemia cells and consequently potentiate the inhibition of growth and cell cycle progression of these cells. We found that overexpression of PTEN with the adenoviral vector Ad/PTEN caused growth arrest at the G1 phase of the cell cycle of HL-60 cells. HL-60 cells treated with either a PPARgamma ligand (ciglitazone) or a RAR ligand (all-trans retinoic acid [ATRA]) up-regulated PTEN in HL-60 cells. The 2 compounds in combination showed synergistic effects on PTEN expression at the protein and messenger RNA levels. Moreover, the combination of ciglitazone and ATRA synergistically reduced cell growth rates and cell cycle arrest at the G1 phase. Our results suggest that, PPARgamma and RAR play an important role in controlling the growth of leukemia cells via the up-regulation of PTEN.     

Acetaldehyde inhibits the formation of retinoic acid from retinal in the rat esophagus

OBJECTIVE: It has already been demonstrated that the rat esophagus produces retinoic acid from retinol. In this study, this process is further characterized and the effect of acetaldehyde examined to elucidate the possible mechanisms behind the epidemiological evidence that the incidence of esophageal cancer is higher in alcoholics. MATERIAL AND METHODS: Rat esophageal samples were incubated with all-trans retinal and newly formed all-trans retinoic acid (ATRA) was quantified using high-performance liquid chromatography (HPLC). Furthermore, beta-nicotinamide adenine dinucleotide (NAD)-dependent acetaldehyde oxidation by the rat esophagus was examined by tracing NAD reduction using a spectrophotometer. RESULTS: Rat esophageal samples produced ATRA from all-trans retinal in a NAD-dependent manner and the potential was significantly attenuated by phenetyl isothiocynate, an ALDH inhibitor, or acetaldehyde depending on the concentration used. Rat esophageal samples also oxidized acetaldehyde of various concentrations NAD dependently. The ATRA formation potential that was temporarily inhibited by acetaldehyde was recovered to the control level by dialysis when the specimen was incubated with up to 50 microM of acetaldehyde. CONCLUSIONS: The rat esophagus produces retinoic acid from retinal. An ALDH isoform(s) is responsible for this process and physiological concentration of acetaldehyde hampers the process, probably in a competitive manner. Since the disturbance of retinoic acid supply has been implicated in carcinogenicity, this finding may, at least in part, explain the high incidence of esophageal cancer in alcoholics, especially in those with inactive ALDH 2 whose blood acetaldehyde levels become higher than those with active ALDH 2.     

DNA methylation-independent loss of RARA gene expression in acute myeloid leukemia

The retinoic acid receptor (RAR) alpha gene (RARA) encodes 2 major isoforms and mediates positive effects of all-trans retinoic acid (ATRA) on myelomonocytic differentiation. Expression of the ATRA-inducible (RARalpha2) isoform increases with myelomonocytic differentiation and appears to be down-regulated in many acute myeloid leukemia (AML) cell lines. Here, we demonstrate that relative to normal myeloid stem/progenitor cells, RARalpha2 expression is dramatically reduced in primary AML blasts. Expression of the RARalpha1 isoform is also significantly reduced in primary AML cells, but not in AML cell lines. Although the promoters directing expression of RARalpha1 and RARalpha2 are respectively unmethylated and methylated in AML cell lines, these regulatory regions are unmethylated in all the AML patient cell samples analyzed. Moreover, in primary AML cells, histones associated with the RARalpha2 promoter possessed diminished levels of H3 acetylation and lysine 4 methylation. These results underscore the complexities of the mechanisms responsible for deregulation of gene expression in AML and support the notion that diminished RARA expression contributes to leukemogenesis.