Humans ; Leukemia, Promyelocytic, Acute ; genetics ; Oncogene Proteins, Fusion ; genetics ; Receptors, Retinoic Acid ; genetics ; Retinoic Acid Receptor alpha ; STAT5 Transcription Factor ; genetics

OBJECTIVETo investigate the effect of retinoic acid on the differentiation and maturation of B cells from lymph node of children, and relational changes of the expression levels of retinoic acid receptor genes.

METHODSTwenty-four patients with digestive tract malformation underwent surgical operation in the surgical ward of our hospital. They were divided into 3 groups according to age: < 1 yr, 1 - 3 yr, -5 yr, 8 cases in each age group. The lymph nodes in the margin of excised tissues were obtained. The cells separated from lymph nodes were cultured in vitro. The cells were divided into 5 groups: Retinoic acid (RA), RA plus Ro41-5253, RA receptor antagonist (RA+Ro), lipopolysaccharide (LPS), lipopolysaccharide plus RA (LPS+RA) and control, and were subjected to the corresponding treatments. After 24 h and 48 h of cell culture, the surface markers on the B cells were detected by flow cytometer to observe the maturation and activation of B cells. The expression levels of retinoic acid receptor genes were quantitatively analyzed by RT- fluorescent quantitative PCR.

RESULTSAfter culture, in < 1 yr group, the number of the mature B cells (IgM(+)IgD(+)CD25(-)) in the RA group was significantly higher than that in the control (24 h: 23% +/- 5% vs. 17% +/- 3%; 48 h: 28% +/- 6% vs. 22% +/- 4%) (P < 0.05), and that in the RA + RO group was not significantly different from that in the control (24 h: 16% +/- 4%; 48 h: 20% +/- 9%) (P > 0.05). The number of activated B cells (IgM(+)CD25(+)) in the LPS + RA group was obviously higher than that in the LPS group (24 h: 82% +/- 10% vs. 76% +/- 8%; 48 h: 83% +/- 8% vs. 78% +/- 10%)(P < 0.05). The level of RARalpha gene expression of B cells (lg copies/50 ng RNA) in the RA group was significantly higher than that in the control (24 h: 7.03 +/- 1.36 vs. 5.79 +/- 2.05; 48 h: 7.91 +/- 1.60 vs. 6.21 +/- 1.88) (P < 0.05), and that in the LPS + RA group was significantly higher than that in the LPS group (24 h: 7.29 +/- 1.53 vs. 5.98 +/- 1.48; 48 h: 7.83 +/- 1.66 vs. 5.79 +/- 2.36)(P < 0.05). In 1 - 3 yr group the changes of maturation and activation of B cells in the lymph nodes were the same as the < 1 yr group. In -5 yr group, such changes were not significant.

CONCLUSIONRA can promote the development of B cell from lymph node in vitro culture in retinoic acid receptor genes may take part in the ontogenesis of B cell, and play a key role in the regulation of retinoic acid.

B-Lymphocytes ; drug effects ; physiology ; Cells, Cultured ; Child, Preschool ; Female ; Humans ; Infant ; Lymph Nodes ; drug effects ; Lymphocyte Activation ; drug effects ; Male ; Receptors, Retinoic Acid ; genetics ; Retinoic Acid Receptor alpha ; Tretinoin ; pharmacology

OBJECTIVEInactivating mutations of DAX-1 give rise to the X-linked form of adrenal hypoplasia congenita (AHC). Affected individuals are at risk of early postnatal Addisonian crisis, but the variable phenotypic expression of DAX-1 insufficiency renders this diagnosis challenging. This study aimed to understand the clinical features and identify DAX-1 gene mutation of the affected individuals and their relatives in a Chinese adrenal hypoplasia congenita kindred.

METHODSThe proband was diagnosed as adrenal insufficiency shortly after birth and his elder cousin was also diagnosed as having this disease at the age of about 8 years. Clinical data were obtained from 2 affected individuals when they were hospitalized into the department of pediatrics, Ruijin Hospital in 2006; 20 peripheral blood samples were obtained from the affected individuals and their relatives; exons in DAX-1 gene were amplified, and PCR product was purified and sequenced directly for analyzing mutation.

RESULTSA novel hemizygous mutation (T785C) was found in DAX-1 gene in both patients. Some clinical features such as the age of onset were different although these 2 patients carried the same mutation. There were 5 carriers of this mutation in the patients' maternal pedigree.

CONCLUSIONThe results suggested that adrenal hypoplasia congenita in this kindred was caused by a novel mutation (T785C) in DAX-1 gene, and the same mutation can give rise to the variable phenotype.

Adrenal Hyperplasia, Congenital ; genetics ; Asian Continental Ancestry Group ; genetics ; Child ; DAX-1 Orphan Nuclear Receptor ; genetics ; Genetic Diseases, X-Linked ; genetics ; Humans ; Male ; Mutation ; Pedigree ; Receptors, Retinoic Acid ; genetics ; Repressor Proteins ; genetics

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

OBJECTIVETo study the effect of retinoid kappa receptor alpha (RXRalpha) transfection plus treatment with the RXRalpha ligand, 9-cis-RA, on the proliferation and phenotype of platelet-derived growth factor (PDGF) activated hepatic stellate cells (HSCs).

METHODSPDGF activated rat hepatic stellate cells were transfected with eukaryotic expression vector pcDNA3.1- human RXRalpha, and confirmed by Western blot. Proliferation of transfected HSC was assayed by bromodeoxyuridine (BrdU) incorporation as well as MTT, and the phenotype (alpha-smooth muscle actin, desmin) was observed by immunocytochemistry with image analysis.

RESULTSTransfection of the RXRalpha gene and treatment with ligand 9-cis-RA of PDGF-activated HSCs extended the increased expression of RXRalpha protein for at least 168 hours. Cell proliferation and expressions of alpha- smooth muscle actin (alpha-SMA) and desmin were blocked, compared with groups of sham-transfected, PDGF-activated, no transfection, no ligand treatment, and irrelevant ligand treated HSCs.

CONCLUSIONTransfection with the RXRalpha gene followed by 9-cis-RA ligand treatment will inhibit the proliferation and reverse the phenotype of activated HSC.

Animals ; Cell Division ; Cells, Cultured ; Liver ; cytology ; Liver Cirrhosis ; etiology ; Male ; Phenotype ; Platelet-Derived Growth Factor ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, Retinoic Acid ; genetics ; physiology ; Retinoid X Receptors ; Transcription Factors ; genetics ; physiology ; Transfection

To evaluate the relation of PML/RARalpha isoforms in adult APL patients to clinical therapy and prognosis, the picture of blood and bone marrow aspirates for 71 APL patients treated by induction therapy were peridically examined and the different transcripts of PML/RARalpha were assayed by nested RT-PCR. The results showed that the median WBC count (x 10(9)/L) in the patients with the short (S) and variable (V) isoform were significantly higher than that in long (L) isoform. So, the serious bleeding complications early occurred. Relapse risk for patients with S and variable isoforms were higher than that with L isoform. In conclusion, PML/RARalpha isoforms in patients with APL may be the independent prognostic factor.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Female ; Humans ; Leukemia, Promyelocytic, Acute ; blood ; genetics ; pathology ; Leukocyte Count ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; Oncogene Proteins, Fusion ; genetics ; Prognosis ; Protein Isoforms ; genetics ; Receptors, Retinoic Acid ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

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

OBJECTIVETo investigate the correlation between RARbeta gene promoter methylation and P53 gene mutations in non-small cell lung cancer (NSCLC).

METHODSPromoter methylation of RARbeta and P53 mutations of exons 5 through 9 in 198 resected primary NSCLC tissues were determined by methylation-specific PCR and direct sequencing.

RESULTSRARbeta gene promoter methylation and P53 mutation were detected in 58.1% and 36.4% of tumors, respectively. Both were higher in males than in females and in smokers than in nonsmokers. A higher prevalence of RARbeta promoter methylation was found in patients with advanced stage tumors than those with TNM stage I. P53 gene mutations were more frequent in squamous cell carcinoma and adeno-squamous carcinoma than adenocarcinoma. All such differences were statistically significant (P< 0.05). Frequencies of P53 mutations, including G:C>T:A mutations, transversions and missense mutations were significantly higher in tumors with RARbeta methylation than in those without (P< 0.05). A significantly higher prevalence of RARbeta methylation was found in tumors with only G:C>T:A mutation in P53 gene than those without P53 mutations (P< 0.05). This difference (OR=3.737, 95%CI: 1.414-9.873) was still statistically significant (P< 0.05) in smokers (OR=4.020, 95%CI: 1.263-12.800), squamous cell carcinomas (OR=5.480, 95%CI: 1.400-21.446) or patients with advanced tumors (OR=3.446, 95%CI: 1.054-11.267) after adjusting for age and sex.

CONCLUSIONRARbeta methylation is associated with G:C>T:A mutations in P53 gene in NSCLC.

Adult ; Aged ; Base Sequence ; Carcinoma, Non-Small-Cell Lung ; genetics ; pathology ; DNA Methylation ; Female ; Genes, p53 ; Genetic Predisposition to Disease ; Humans ; Lung Neoplasms ; genetics ; pathology ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation ; Promoter Regions, Genetic ; Receptors, Retinoic Acid ; genetics

Our goal is to decipher which DNA sequences are required for tissue-specific expression of epididymal genes. At least 6 epididymis-specific lipocalin genes are known. These are differently regulated and regionalized in the epididymis. Lipocalin 5 (Lcn5 or mE-RABP) and Lipocalin 8 (Lcn8 or mEP17) are homologous genes belonging to the epididymis-specific lipocalin gene cluster. Both the 5 kb promoter fragment of the Lcn5 gene and the 5.3 kb promoter fragment of the Lcn8 gene can direct transgene expression in the epididymis (Lcn5 to the distal caput and Lcn8 to the initial segment), indicating that these promoter fragments contain important cis-regulatory element(s) for epididymis-specific gene expression. To define further the fragments regulating gene expression, the Lcn5 promoter was examined in transgenic mice and immortalized epididymal cell lines. After serial deletion, the 1.8 kb promoter fragment of the Lcn5 gene was sufficient for tissue-specific and region-specific gene expression in transgenic mice. Transient transfection analysis revealed that a transcription factor forkhead box A2 (Foxa2) interacts with androgen receptor and binds to the 100 bp fragment of the Lcn5 promoter between 1.2 kb and 1.3 kb and that Foxa2 expression inhibits androgen-dependent induction of the Lcn5 promoter activity. Immunohistochemistry indicated a restricted expression of Foxa2 in the epididymis where endogenous Lcn5 gene expression is suppressed and that the Foxa2 inhibition of the Lcn5 promoter is consistent with the lack of expression of Lcn5 in the corpus and cauda. Our approach provides a basic strategy for further analysis of the epididymal lipocalin gene regulation and flexible control of epididymal function.
Animals ; Base Sequence ; Carrier Proteins ; genetics ; Epididymis ; physiology ; Hepatocyte Nuclear Factor 3-beta ; genetics ; Humans ; Lipocalins ; Male ; Mice ; Molecular Sequence Data ; Multigene Family ; Promoter Regions, Genetic ; Prostate ; physiology ; Receptors, Retinoic Acid ; genetics ; Retinol-Binding Proteins, Plasma