OBJECTIVETo study the expression of retinoic acid receptor-beta (RAR-beta) mRNA and p16, p53, Ki67 proteins in squamous-cell carcinoma of the esophagus and its precursor lesions in a high risk population.

METHODSA total of 397 tissue specimens were collected from individuals with normal mucosa (NM, n = 25), mild dysplasia (MiD, n = 69), moderate dysplasia (MoD, n = 106), severe dysplasia (SD, n = 51), carcinoma in situ (CIS, n = 78), and squamous-cell carcinoma (SC, n = 68). Expression of RAR-beta mRNA was detected by in situ hybridization, and that of p16, p53 and Ki67 proteins by immunohistochemistry.

RESULTSThe frequencies of RAR-beta mRNA expression in NM, MiD, MoD, SD, CIS and SC were 96.0%, 89.9%, 67.9%, 68.6%, 62.8%, and 63.2%, respectively. The frequencies of p16 expression were 88.0%, 71.0%, 64.2%, 51.0%, 53.8% and 52.9%; those of p53 expression were 4.0%, 39.1%, 57.5%, 52.9%, 67.9% and 69.1%; those of Ki67 expression were 0, 40.6%, 61.3%, 58.8%, 59.0% and 75.0%, respectively.

CONCLUSIONThere are no significant differences in four biomarkers expression between carcinoma of the esophagus and its precursor lesions.

Biomarkers, Tumor ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Esophageal Neoplasms ; metabolism ; Esophagus ; metabolism ; Humans ; Ki-67 Antigen ; metabolism ; Precancerous Conditions ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Retinoic Acid ; biosynthesis ; genetics ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo investigate celecoxib-induced apoptosis of acute promyelocytic leukemia cell line MR2 and related mechanism.

METHODSMR2 cells were treated with celecoxib at different concentrations (0, 20, 40, 80, 120 and 160 micromol/L). The proliferation of MR2 cells was observed by MTT assay and apoptosis was detected by DNA fragmentation analysis and flow cytometry with Annexin V-FITCïPI staining. The expression of survivin and PML/RARalpha mRNA was examined by RT-PCR and nested-PCR, and the protein expression of caspase-3, 9 and PARP was analyzed by Western-blot.

RESULTSAfter treatment with celecoxib the viability of MR2 cells decreased markedly in a dose- and time-dependent manner, and a DNA ladder pattern of internucleosomal fragmentation was observed. The translocation of phosphatidylserine at the outer surface of the cell plasma membrane was induced by celecoxib and its level increased following the augmentation of the drug concentration. The expression of survivin mRNA decreased dramatically while no significant change with PML/RARalpha. Treatment with celecoxib for 24 h resulted in the activation of caspase-3 and 9, cleavage of PARP.

CONCLUSIONCelecoxib could inhibit MR2 cell proliferation by inducing apoptosis, which might be mediated by the caspase-3 and 9 activation and PARP cleavage. Moreover, the down-regulation of survivin may play a certain role in apoptosis of MR2 cells induced by celecoxib.

Apoptosis ; drug effects ; Blotting, Western ; Caspase 3 ; metabolism ; Celecoxib ; Cell Line, Tumor ; Cell Survival ; drug effects ; Collagen Type XI ; metabolism ; Cyclooxygenase 2 Inhibitors ; pharmacology ; Flow Cytometry ; Humans ; Inhibitor of Apoptosis Proteins ; Leukemia, Promyelocytic, Acute ; genetics ; metabolism ; pathology ; Microtubule-Associated Proteins ; genetics ; Neoplasm Proteins ; genetics ; Pyrazoles ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Receptors, Retinoic Acid ; genetics ; Retinoic Acid Receptor alpha ; Reverse Transcriptase Polymerase Chain Reaction ; Sulfonamides ; pharmacology

In order to explore the application of real-time quantitative reverse-transcription polymerase chain reaction (Q-PCR) for detecting PML/RARalpha gene transcripts in patients with acute promyelocytic leukemia (APL), the bone marrow samples from 46 newly diagnosed APL patients were collected for analysis. Three plasmids containing cDNA fragments of the bcr1-, bcr3-form PML/RARalpha and ABL control gene were constructed respectively. The ABI Prism 7500 Sequence Detection System using Taqman fluorogenic probes was used to quantify target gene. PML/RARalpha mRNA was detected by Q-PCR in 46 APL patients and 40 non-APL patients. The normalized quotient (NQ) of PML/RARalpha mRNA was calculated as followings: NQ = PML/RARalpha mRNA copy numbers/ABL mRNA copy numbers. Immunophenotype of acute promyelocytic leukemia was determined by four-color flow cytometry. The results showed that the coefficients of variation (CV) of inter-day assay and intra-day assay by using Q-PCR were 1.58% and 0.88% respectively. Q-PCR could detect reproducibly 5 copies of target gene per 100 ng RNA and no pseudopositive results were found. The median NQ of PML/RARalpha mRNA was 0.450 (0.084 - 1.082) in 46 APL patients. There was no indication of any correlation of PML/RARalpha mRNA type with age, sex, hemoglobin, platelet count, percentage of promyelocytes in bone marrow detected by morphology or flow cytometry, PML/RARalpha NQ, or signs of clinically diagnosed coagulation/bleeding disorders. Compared with bcr1-form cases, bcr3-form cases had more M(3v) phenotype (42.9% vs 9.4%, P = 0.015) and higher WBC count (9.35 x 10(9)/L vs 2.15 x 10(9)/L, P = 0.038). APL cells could be classified into large side scatter population (L-SSC) and non-large side scatter population (NL-SSC) in CD45/SSC histogram of flow cytometry. 87.50% patients with bcr1-form showed L-SSC phenotype and 64.29% patients with bcr3-form showed NL-SSC phenotype. It is concluded that a sensitive Q-PCR method is established. The median NQ of PML/RARalpha mRNA was 0.450 in newly diagnosed APL patients. There was no significant difference about PML/RARalpha mRNA expression of both bcr3-form and bcr1-form APL patients. Type of PML/RARalpha transcripts is related with the morphology and immunophenotype.
Adolescent ; Adult ; Aged ; Child ; Female ; Genes, abl ; genetics ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; genetics ; metabolism ; Male ; Middle Aged ; Oncogene Proteins, Fusion ; analysis ; genetics ; Phenotype ; RNA, Messenger ; analysis ; genetics ; Receptors, Retinoic Acid ; analysis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods

To explore the value of detection of PML/RARalpha gene rearrangement on bone marrow smears (BMS) by dual-color fluorescence in situ hybridization (D-FISH) for the diagnosis of acute promyelocytic leukemia (APL), the locus-specific probes for PML and RARalpha genes labeled directly and respectively by Spectrum Green and Spectrum Orange and the D-FISH technique were used to detect the PML/RARalpha gene rearrangement on BMS in 27 suspected APL patients. The results were compared with that of conventional cytogentics (CCG) and reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that out of 18 newly diagnosed patients 14 were found having t(15;17) translocation by CCG and PML/RARalpha gene rearrangement were confirmed by BMS-D-FISH and RT-PCR. Thus, their APL diagnosis was determined; out of 4 patients in whom t(15;17) translocation was not detected by CCG, one had positive BMS-D-FISH and RT-PCR results, thus, this case was considered as having a cryptic t(15;17) translocation, three had negative BMS-D-FISH and RT-PCR results, thus, they were diagnosed as having acute myeloid leukemia rather than APL. In 9 cases with remission, one case with partial remission was found having t(15;17) translocation by CCG, and he had positive BMS-D-FISH and RT-PCR results, the other 8 patients (6 cases with normal karyotype and 2 cases without CCG examination) displayed different BMS-D-FISH and RT-PCR results: negative in 6 cases, but positive in 2 cases. The 2 cases were believed that they survived with minimal residual disease (MRD). It is concluded that BMS-D-FISH is a sensitive and reliable method for the detection of PML/RARalpha rearrangement. It is helpful for diagnosing APL and monitoring its MRD, and especially fit to those patients presenting a cryptic translocation or with failed cytogenetics, lacking suitable material for RT-PCR, as well as needing retrospective study.
Adolescent ; Adult ; Bone Marrow Cells ; metabolism ; Female ; Gene Rearrangement ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Leukemia, Promyelocytic, Acute ; diagnosis ; genetics ; Male ; Middle Aged ; Oncogene Proteins, Fusion ; genetics ; Receptors, Retinoic Acid ; genetics ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity

To study the coumarins of Anemone raddeana Regel, the compounds were separated by silica gel column chromatography and HPLC. Their structures were identified by their physicochemical property and spectral analysis. Two new compounds were isolated and identified as 4, 7-dimethoxyl-5-methyl-6-hydroxy coumarin (1) and 4, 7-dimethoxyl-5-formyl-6-hydroxycoumarin (2). The bioassays indicated that compounds 1 and 2 could significantly inhibit the proliferation of cancer cell, and showed the agonist effect on the transactivity of retinoic acid receptor-alpha (RARalpha). In addition, the two compounds had inhibitory effect against human leukocyte elastase (HLE).
Anemone ; chemistry ; Antineoplastic Agents, Phytogenic ; chemistry ; isolation & purification ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Coumarins ; chemistry ; isolation & purification ; pharmacology ; Humans ; Inhibitory Concentration 50 ; Leukocyte Elastase ; metabolism ; Molecular Structure ; Plants, Medicinal ; chemistry ; Receptors, Retinoic Acid ; genetics ; metabolism ; Retinoic Acid Receptor alpha ; Rhizome ; chemistry ; Transcriptional Activation

INTRODUCTIONIgA nephropathy is a disease where the pathogenesis is still poorly understood. Deoxyribonucleic acid (DNA) microarray technique allows tens of thousands of gene expressions to be examined at the same time. Commercial availability of microarray genechips has made this powerful tool accessible for wider utilisation in the study of diseases.

MATERIALS AND METHODSSeven patients with IgA nephropathy, 6 with minimal change nephrotic syndrome (MCNS) as patient controls and 7 normal healthy subjects were screened for the differential expression of genes, genome-wide. The Human Genome U133 Plus 2.0 Arrays (Affymetrix, USA) were used to quantitate the differential expression of 38,500 well-characterised human genes.

RESULTSA total of 7761 gene expressions were identified that have an IgAN/Normal gene expression ratio of 0.06-fold to 5.58-fold. About 35% of the altered gene expressions have no gene title or just a hypothetical protein label such as FLJ30679. Most of the remaining 65% are identified proteins where their importance to IgAN is not immediately apparent at this time. Among the 30 most upregulated and 30 most downregulated genes are Urotensin 2 (upregulated 3.09-fold, P <0.05) and Fatty-acid binding protein 6 (downregulated to 0.12-fold, P <0.05). Retinoic acid receptor alpha (vitamin A receptor) was also found downregulated to 0.41-fold (P <0.005). Taqman realtime polymerase chain reaction (PCR) for urotensin 2 and retinoic acid receptor alpha (RARA) were performed on 20 patients with IgA nephropathy and 11 with Minimal Change Disease and the data correlated with various clinical indices.

CONCLUSIONSThe findings suggest that there may be a therapeutic role for retinoic acid receptor alpha (RARA) in IgA nephropathy and a clinical monitoring role for Urotensin 2 in Minimal Change Disease.

Adult ; Aged ; Case-Control Studies ; Female ; Gene Expression ; Gene Expression Regulation ; Genome-Wide Association Study ; Glomerulonephritis, IGA ; genetics ; metabolism ; pathology ; Humans ; Immunoglobulin A ; genetics ; metabolism ; Male ; Middle Aged ; Nephrosis, Lipoid ; genetics ; metabolism ; pathology ; Oligonucleotide Array Sequence Analysis ; Polymerase Chain Reaction ; Receptors, G-Protein-Coupled ; genetics ; metabolism ; Receptors, Retinoic Acid ; genetics ; metabolism ; Tretinoin ; metabolism

In response to viral infection, RIG-I-like RNA helicases detect viral RNA and signal through the mitochondrial adapter protein VISA. VISA activation leads to rapid activation of transcription factors IRF3 and NF-κB, which collaborate to induce transcription of type I interferon (IFN) genes and cellular antiviral response. It has been demonstrated that VISA is activated by forming prion-like aggregates. However, how this process is regulated remains unknown. Here we show that overexpression of HSC71 resulted in potent inhibition of virus-triggered transcription of IFNB1 gene and cellular antiviral response. Consistently, knockdown of HSC71 had opposite effects. HSC71 interacted with VISA, and negatively regulated virus-triggered VISA aggregation. These findings suggest that HSC71 functions as a check against VISA-mediated antiviral response.
Adaptor Proteins, Signal Transducing ; biosynthesis ; chemistry ; genetics ; metabolism ; Cell Aggregation ; genetics ; GPI-Linked Proteins ; metabolism ; Gene Knockdown Techniques ; HEK293 Cells ; HSC70 Heat-Shock Proteins ; genetics ; metabolism ; Heat-Shock Response ; genetics ; Humans ; Interferon Regulatory Factor-3 ; genetics ; metabolism ; Interferon-beta ; genetics ; NF-kappa B ; genetics ; Prions ; metabolism ; Receptors, Retinoic Acid ; metabolism ; Viruses ; drug effects ; metabolism ; pathogenicity

In general, a 2-yr disease-free duration is recommended before kidney transplantation (KT) in end-stage renal disease (ESRD) patients who also have acute leukemia. However, the optimal disease-free interval has not been specified for all subtypes of acute leukemia. Among these subtypes, acute promyelocytic leukemia (APL) shows a favorable prognosis and low relapse rate compared to other types of leukemia. We here report KT after complete remission (CR) of APL in an ESRD patient. Irreversible kidney injury developed in a 23-yr-old man with APL. First, we induced CR and subsequently performed KT 7 months after the achievement of CR. The patient's clinical course after KT was favorable, without allograft rejection or relapse of APL up to1 yr after KT. On the basis of our clinical experience, it is suggested that a long wait may not be necessary before KT in patients with ESRD and APL.
Adult ; Antineoplastic Agents/therapeutic use ; Arsenicals/therapeutic use ; Bone Marrow Cells/pathology ; Humans ; Kidney Failure, Chronic/*therapy/ultrasonography ; *Kidney Transplantation ; Leukemia, Promyelocytic, Acute/*diagnosis/drug therapy ; Male ; Oxides/therapeutic use ; Receptors, Retinoic Acid/genetics/metabolism ; Remission Induction

Mammals have two major isoforms of acetyl-CoA carboxyase (ACC). The 275 kDa beta-form (ACC beta) is predominantly in heart and skeletal muscle while the 265 kDa alpha-form (ACC alpha) is the major isoform in lipogenic tissues such as liver and adipose tissue. ACC alpha is thought to control fatty acid oxidation by means of the ability of malonyl-CoA to inhibit carnitine palmitoyl-CoA transferase-1 (CPT-1), which is a rate-limiting enzyme of fatty acid oxidation in mitochondria. Previously, it was reported that MyoD and other muscle regulating factors (MRFs) up-regulate the expression of ACC beta by interactions between these factors and several cis-elements of ACC beta promoter. We described here that ACC beta expression mediated by MRFs is regulated by retinoic acids. Endogenous expression of ACCb in differentiated H9C2 myotube was significantly increased by retinoic acid treatment. However, on transient transfection assay in H9C2 myoblast, ACC beta promoter activity was suppressed by RXRa and more severely by RAR alpha. These effects on ACCb expression in myoblasts and myotubes by RXR alpha and RAR alpha seem to be mediated by their interactions with MRFs because no consensus sequence for RXR alpha and RAR alpha has been found in ACC beta promoter and retinoic acid receptors did not affect this promoter activities by itself. In transient transfection in NIH3T3 fibroblast, the activation of ACC beta promoter by MyoD, main MRF in myoblast, was significantly suppressed by RAR alpha and to a less extent by RXR alpha while the RXR alpha drastically augmented the activation by MRF4, major MRF in myotube. These results explained that retinoic acids differentially affected the action of MRFs according to their types and RXR alpha specially elevates the expression of muscle specific genes by stimulating the action of MRF4.
3T3 Cells ; Acetyl-CoA Carboxylase/genetics/*metabolism ; Animals ; Cell Differentiation ; Cells, Cultured ; Gene Expression Regulation, Enzymologic/drug effects ; Mice ; MyoD Protein/*metabolism ; Myoblasts/drug effects/metabolism ; Myogenic Regulatory Factors/*metabolism ; Promoter Regions (Genetics)/drug effects ; Receptors, Retinoic Acid/genetics/*metabolism ; Trans-Activation (Genetics) ; Transcription Factors/genetics/*metabolism ; Tretinoin/pharmacology

The growth inhibitory effects of four retinoic acid (RA) derivatives, 9-cis RA, 13-cis RA, N-(4-hydroxyphenyl) retinamide (4-HPR), and all-trans retinoic acid (ATRA) were compared. In addition, the effects of various combinations of these four agents were examined on non-small cell lung carcinoma (NSCLC) cell-lines, and on the expressions of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) on these cells. At the clinically achievable concentration of 1 micrometer, only 4-HPR inhibited the growths of H1299 and H460 cells-lines. However, retinoic acid receptor beta(RAR beta) expression was up-regulated on H460 and H1299 cells treated with 1 micrometer of ATRA, 13-cis RA, or 9-cis RA. All NSCLC cell lines showed growth inhibition when exposed sequentially to 1 micrometer ATRA and 0.1 micrometer 4-HPR. In particular, sequential treatment with 1 micrometer ATRA or 13-cis RA and 4-HPR markedly inhibited H1703 cell growth; these cells exhibited no basal RAR beta expression and were refractory to 4-HPR. However, in NSCLC cell lines that expressed RAR beta, the expressional levels of RAR beta were up-regulated by ATRA alone and by sequential treatment with ATRA and 4-HPR. 4-HPR was found to be the most active of the four agents in terms of NSCLC growth-inhibition. Moreover, sequential treatments with ATRA or 13-cis RA followed by 4-HPR were found to have synergistic growth-inhibitory effects and to regulate RAR expression.
Base Sequence ; Carcinoma, Non-Small-Cell Lung/*drug therapy/genetics/metabolism ; Cell Line, Tumor ; DNA Primers/genetics ; Drug Therapy, Combination ; Fenretinide/administration & dosage ; Gene Expression/drug effects ; Humans ; Isotretinoin/administration & dosage ; Lung Neoplasms/*drug therapy/genetics/metabolism ; Receptors, Retinoic Acid/genetics ; Retinoid X Receptors/genetics ; Tretinoin/administration & dosage/*analogs & derivatives