OBJECTIVETo screen the protein interacting with retinoic acid receptor variant protein (RAR alpha-V) via the yeast two-hybrid technique (YTHT) and to find out the targets protein and study its biological function.

METHODSThe bait vector of pGBKT7-RAR alpha-V was constructed for screening proteins interacting with RAR alpha-V in K562 cell cDNA expression library via YTHT. The protein-protein interaction was confirmed with re-transformation in yeast and GST pull-down in vitro.

RESULTSThe bait vector was successfully constructed without toxicity, leakage and self-activation. Sixteen proteins were screened by YTHT and eight positive clones were identified by re-transformation in yeast. The interaction between RAR alpha-V and JTV-1 was confirmed by GST pull-down in vitro.

CONCLUSIONSThere are some kinds of proteins interacting with RAR alpha-V in cell. The biological dysfunction caused by certain protein-protein interaction may be involved in the pathogenesis of leukemia.

Gene Library ; Humans ; K562 Cells ; Protein Interaction Mapping ; Receptors, Retinoic Acid ; metabolism ; Retinoic Acid Receptor alpha ; Two-Hybrid System Techniques

OBJECTIVETo investigate the changes in cell proliferation and retinoic acid receptor gamma (RARgamma) mRNA expression in normal human keratinocytes after acitretin treatment and/or narrow-band ultraviolet-B irradiation.

METHODSNormal human keratinocytes were exposed to irradiation with 100 mJ/cm square NB-UVB and/or subsequent 12-hour incubation with 1x10(-6) mol/L acitretin, and the expression of RARgamma mRNA in the cells was examined using RT-PCR and real-time quantitative RT-PCR.

RESULTSA 0.9- and a 2.3-fold increase in RARgamma mRNA expression was induced in the cells by exposure to 100 mJ/cm square NB-UVB and 10(-6) mol/L acitretin, respectively, and the expression was synergistically enhanced by 2.8-fold after their combined treatment.

CONCLUSIONUpregulated expression of RARgamma mRNA can be associated with keratinocyte growth inhibition after treatment with acitretin and NB-UVB irradiation.

Acitretin ; pharmacology ; Cells, Cultured ; Humans ; Keratinocytes ; drug effects ; radiation effects ; RNA, Messenger ; metabolism ; Receptors, Retinoic Acid ; metabolism ; Ultraviolet Rays

OBJECTIVETo explore the mechanism of Alpha-fetoprotein (AFP) effects on hepatocellular carcinoma cells (HCC) resistances apoptosis induced by tumor necrosis factor-related apoptosis inducing-ligand (TRAIL).

METHODSThe expressed alteration of TRAIL receptor-2 (DR5) after the human hepatoma cells line Bel 7402 (AFP-producing) and HLE cells (non-AFP producing) were treated with all trans retinoic acid (ATRA) were determined by Western blot; Interaction of AFP with RAR-beta was analyzed by co-immunoprecipitation (Co-IP); Laser confocal microscopy was used to observe co-localization of AFP and RAR-beta; Short small RNA interfering (RNAi) was applied to knock down the expression of AFP in Bel 7402 cells; The full AFP gene cDNA was inserted into pcDNA3.1 vector and constructed the expressed vector of AFP (named pcDNA3.1-afp); The growth of hepatoma cells was analyzed by MTT.

RESULTSBel 7402 and HLE cells expressed DR5, lowed dosage of ATRA (40mumol/L) had no influence on the expression of DR5 in Bel 7402 cells, but ATRA (160mumol/L) could inhibit the expression of AFP and promote the expression of DR5 significantly; Co-IP indicated that AFP had a property for interacting with RAR-beta; The results also demonstrated AFP co-localization with RAR-beta in cytoplasm of Bel 7202 cells; The expression of DR5 was enhanced while the expression of AFP was knocked down by RNAi. pcDNA3.1-afp vector was transfected into HLE cells, the growth of HLE cells were stimulated and TRAIL cytotoxicity of HLE cells were reduced. But when the expression of AFP was knocked down the sensitivity of Bel 7402 cells to TRAIL was enhanced.

CONCLUSIONSThese data provided that AFP had a capability to interact with RAR-beta and suppressed the expression of DR5. AFP could play pivotal role on hepatoma cells resistance-induced apoptosis by TRAIL.

Apoptosis ; Cell Line, Tumor ; metabolism ; Humans ; Receptors, Retinoic Acid ; metabolism ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Tretinoin ; pharmacology ; alpha-Fetoproteins ; metabolism

BACKGROUND AND OBJECTIVES: All-trans retinoic acid (RA) is a form of vitamin A analogue that has shown chemopreventive activities in many types of malignancy with the properties that regulate cellular differentiations and suppress malignant proliferations. It is thought that the catabolism of RA is mediated by cytochrome P450RAI (CYP26) and its absorption, effects and catabolism are related to cellular retinol binding proteins (CRBP-I and II) and cellular retinoic acid binding proteins (CRABP-I and II). With eight different squamous cell carcinoma cell lines (AMC-HN-1~-8), we investigated the effects of RA and roles of these proteins in the metabolism and regulatory activity of RA. MATERIALS AND METHODS: Survival fractions of eight AMC-HN cells were analyzed six days after the treating them with 1 nM of RA. Reverse transcription-polymerase chain reactions (RT-PCR) were performed before and 24 hours after the load of 1 nM of RA to detect the expressions of CRBP-I, CRABP-I, CRABP-II, and CYP26. RESULTS: Resistances to RA were detected in AMC-HN-1, -2, -5, and -6 cell lines after RA treatment, and AMC-HN-3, -4, -7, and -8 cell lines showed sensitive responses to RA. Before the addition of RA, expressions of CYP26 were detected in AMC-HN-1, -2, -4, -5, -6, and -7 cell lines and CRBP-I was expressed in AMC-HN-3, 4, 5, 7, and -8. After RA addition, the expressions of CYP26 were enhanced in AMC-HN-2, -5, -6, and -7. In six of eight cell lines, CRABP-I was suppressed and CRABP-II was enhanced after RA treatment. CONCLUSION: These results suggest that CYP26 has a direct correlation with the cellular metabolism of RA in the head and neck squamous cell carcinomas and that CRABP-I and CRABP-II have distinct roles in the regulatory effects of RA. CRBP-I might be an indicator that implies the responsiveness to RA.
Absorption ; Carcinoma, Squamous Cell* ; Cell Line ; Cytochromes ; Head* ; Metabolism* ; Neck* ; Receptors, Retinoic Acid ; Retinol-Binding Proteins, Cellular ; Tretinoin* ; Vitamin A

This study was aimed to investigate the mechanism of all-trans retinoic acid (ATRA) up-regulating apelin expression in vascular smooth muscle cells (VSMCs). The effect of ATRA on apelin expression in the VSMCs was investigated by RT-PCR, real-time PCR and Western blot analysis. To further define whether retinoic acid receptor α (RARα) mediated the induction of apelin by ATRA, endogenous RARα was down regulated by transfection of siRNA against RARα (si-RARα) or RARα was over-expressed by infection of the adenovirus vector pAd-GFP-RARα in the VSMCs. The results showed that ATRA significantly induced apelin expression in a time- and dose-dependent manner in the VSMCs. Although RARα expression was increased in a time-dependent manner, the expressions of RARβ and RARγ were little changed by the ATRA treatment. When VSMCs were treated with a RARα antagonist Ro 41-5253 prior to the addition of ATRA, or si-RARα was used to down regulate endogenous RARα expression, the blockade of RARα signaling partially reduced the response of apelin to ATRA. Moreover, RARα over-expression, induced by infection of pAd-GFP-RARα, further increased the induction of apelin by ATRA. In conclusion, ATRA may up-regulate apelin expression in VSMCs, and the mechanism may be RARα dependent.
Benzoates ; Chromans ; Gene Expression Regulation ; Intercellular Signaling Peptides and Proteins ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptors, Retinoic Acid ; metabolism ; Retinoic Acid Receptor alpha ; Signal Transduction ; Transfection ; Tretinoin ; metabolism ; Up-Regulation

OBJECTIVETo investigate the effect of all-trans retinoid acid (ATRA) and granulocyte colony-stimulating factor (G-CSF) on the growth, apoptosis, differentiation and expression of RARα2 of myeloma cells.

METHODSMyeloma cell lines OPM2 (RARα2 positive) and U266 (RARα2 negative) were treated with ATRA in the presence or absence of G-CSF. The cells were divided into 6 groups: control groups, G-CSF groups (treated with 1000 U/ml and 2000 U/ml), ATRA groups (treated with 1.0 μmol/L ATRA) and combined groups (treated with 1000 U/mL or 2000 U/mL G-CSF plus 1.0 μmol/L ATRA). The cell viability, growth and apoptosis were examined by MTT method, inverted microscopy and Annexin-V/PI staining, respectively; RARα2 expression was detected by reverse transcription PCR; morphology change was evaluated by Wright-Giemsa staining; CD49e expression were analyzed by flow cytometry.

RESULTSThe proliferation of OPM2 cells was inhibited by ATRA treatment (P<0.05) . The growth inhibition rates in combined groups were higher than corresponding single ATRA groups (P<0.05). However, the above effects in U266 cells were not significant (P >0.05). The OPM2 cell stained by Wright-Giemsa in ATRA groups showed that the cell nucleus became smaller, chromatin condensed, number of nucleolus reduced, the volume of cytoplasm increased and the cytoplasm became dark blue. Expression rates of CD49e were low in both U266 and OPM2 cells. Expression of RARα2 in OPM2 cells of combination groups were higher than those of control group and corresponding single groups (P<0.05); and there was no significant difference between control group and G-CSF groups (P>0.05). Expression of RARα2 in U266 cells of control group and G-CSF groups was not detected; and ATRA groups and combination groups had weak expression.

CONCLUSIONATRA can induce proliferation inhibition in RARα2-expressing myeloma cells, and it may also play a certain role in promoting differentiation of RARα2 positive myeloma cells.

Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Humans ; Multiple Myeloma ; metabolism ; pathology ; Receptors, Retinoic Acid ; metabolism ; Retinoic Acid Receptor alpha ; Tretinoin ; pharmacology

OBJECTIVETo investigate the expression and its significance of retinoic acid receptor-beta (RAR-beta) in colorectal cancer.

METHODSRAR-beta was detected by immunohistochemistry methods and carcino-embryonic antigen (CEA) was tested by chemiluminescence immunoassay methods in normal tissues, paracancerous tissues and colorectal cancer tissues of 60 patients with colorectal cancer treated from January 2006 to January 2007. Above-mentioned data, together with the clinicopathological data of these 60 patients, were analyzed to figure out the expression and its significance of RAR-beta in colorectal cancer.

RESULTSThe expression rate of RAR-beta in tumor tissues (48%) was significantly lower than those in both normal tissues (87%) and paracancerous tissues (87%) (P < 0.05). And its expression was also significant lower in patients with lymph node metastasis (32%) and patients with advanced cancer (TNM stage III and IV) (29%) than in those without lymph nodes metastasis (60%) and those with early stage cancer (stage I and II) (69%). There was no significant differences among well, mildly and poorly differentiated cancer tissues. The CEA level rose in 20 patients, and its rising rate was remarkably higher in patients with lymph node metastasis (48%) and in patients with advanced cancer (52%) than those without lymph node metastasis (23%) and in early stage(14%).

CONCLUSIONSThe expression of RAR-beta decreases significantly in cancer tissues in patients with colorectal cancer, which may be related to the carcinogenesis of colorectal cancer; and its decreasing degree is correlated negatively with the lymph node metastasis and advanced clinicopathological stage. The expression level of RAR-beta may be a new prognostic indication of patients with colorectal cancer.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Colorectal Neoplasms ; metabolism ; pathology ; Female ; Humans ; Male ; Middle Aged ; Receptors, Retinoic Acid ; metabolism ; Young Adult

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

Retinoic acid receptor- (RAR-beta) is induced by and mediates the growth-inhibitory and apoptotic effects of retinoic acid (RA), suggesting that loss of RAR-betaexpression may be one of the critical events involved in the carcinogenesis/ progression of non-small cell lung cancer (NSCLC) and in the responsiveness to retinoid chemotherapy. However, recent contradictory reports that the expression of RAR-beta is associated with poor clinical outcome, and the fact that treatment of serum-deprived type 2 alveolar cells with RA leads to a stimulation of cell proliferation, require the verification of RAR-beta as a biomarker of chemoprevention or prognosis. The expression status of RAR-beta in cancer cells and adjacent normal appearing bronchial epithelium from 39 patients, diagnosed as stage I NSCLC and undergone a curative lung resection, was analyzed in paraffin-embedded tissue sections by IHC staining. The normal appearing bronchial epithelium of 14 out of 33 (42.4%) specimens expressed RAR-beta, whereas 22 out of the 39 (56.4%) stage I NSCLC specimens expressed RAR-beta. RAR-beta was more frequently expressed in the adenocarcinoma (72.7%) than in the squamous cell carcinoma (31.3%) (p=0.026). Neither the expression status in normal appearing adjacent tissue nor that in the tumor tissue had prognostic implications. The higher expression of RAR-beta in cancer tissue, the focal and uneven distribution in normal appearing adjacent bronchial epithelium, and inconsistency with the corresponding tumor tissue, suggest that the expression status of RAR-beta as a biomarker for chemoprevention/early diagnosis or the prognosis of NSCLC requires further consideration.
Adult ; Aged ; Bronchi/metabolism/pathology ; Carcinoma, Non-Small-Cell Lung/*metabolism/pathology ; Down-Regulation ; Female ; Human ; Immunohistochemistry ; Lung Neoplasms/*metabolism/pathology ; Male ; Middle Aged ; Neoplasm Staging ; Receptors, Retinoic Acid/*metabolism ; Respiratory Mucosa/*pathology ; Tumor Markers, Biological/*metabolism