OBJECTIVETo construct a subtractive cDNA library of genes transactivated by hepatitis B virus X protein (HBX) using suppression subtractive hybridization (SSH) technique and to clone genes associated with HBX transactivating function.

METHODSThe mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-X and pcDNA3.1(-) empty vector respectively, then cDNA was synthesized. After restriction enzyme RsaI digestion, a number of small size cDNA was obtained. Then tester cDNA was subdivided into two portions and each was ligated with different cDNA adaptor. After tester cDNA was hybridized with driver cDNA twice and underwent nested polymerase chain reaction (PCR) twice the production was subcloned into T/A plasmid vectors to set up the subtractive cDNA library. Amplification of the library was carried out with E. coli strain JM109, some cDNA was sequenced and analyzed in GenBank with Blast.

RESULTSThe subtractive cDNA library of genes transactivated by HBX was constructed. The amplified library contained 85 positive clones, and colony PCR showed that these clones contained 200-1000 bp inserts. 65 clones were analyzed by sequencing and bioinformatics, which suggested nineteen known genes and fifteen genes with unknown function.

CONCLUSIONA subtractive cDNA library of genes transactivated by HBX using SSH technique has been constructed successfully, which may bring some new clues for studying the biological functions of HBX and the pathogenesis of hepatoma.

Cloning, Molecular ; Gene Library ; RNA, Messenger ; analysis ; Trans-Activators ; physiology ; Transcriptional Activation

To determine the feasibility of a nonradioactive electrophoresis mobility shift assay for detecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus target sequence of NF-kappa B were labelled with DIG by terminal transferase. After nuclear protein stimulated with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDTC) electrophoresed on 8% nondenaturing poliacrylamide gel together with oligeonucleotide probe, they were electro-blotted nylon membrane positively charged. Anti-DIG-AP antibody catalyzed chemiluminescent substrate CSPD to image on X-film. The results showed that nuclear proteins binded specifically to the NF-kappa B consensus sequence in the EMSA by chemiluminescent technique method and the activity of NF-kappa B in PMA group was more than that in PMA + PDTC group. It is suggested that detection of NF-kappa B by EMSA with chemiluminescent technique is feasible and simple, which can be performed in ordinary laboratories.
Chemiluminescent Measurements ; DNA-Binding Proteins/*analysis ; DNA-Binding Proteins/genetics ; Electrophoretic Mobility Shift Assay ; NF-kappa B/*analysis ; NF-kappa B/genetics ; NF-kappa B/metabolism ; Rats, Sprague-Dawley ; *Trans-Activation (Genetics) ; Trans-Activators/analysis ; Trans-Activators/genetics ; *Transcription, Genetic

Loss of the cell adhesion molecule E-cadherin is suggested to promote tumor invasion and distant metastasis in tumor development. Recently, it has been proposed that E-cadherin function requires its linkage to the cytoskeleton through catenins. We evaluated the expression of E-cadherin and alpha-, beta-, gamma-catenins in tissues of human endometrial carcinoma, analyzed the patterns of cell adhesion molecules' expression in endometrial carcinoma and investigated the relationship between the statuses of cell adhesion molecules and various clinicopathological factors. This study investigated the immunohistochemical expression of E-cadherin and alpha-, beta-, gamma-catenins in 33 paraffin embedded formalin fixed tissues of endometrial carcinomas. Aberrant E-cadherin, and alpha-, beta-, gamma-catenin expression was observed in 33.3 (11 of 33), 27.3 (9 of 33), 18.2 (6 of 33), and 51.5 (17 of 33) % of the specimens, respectively. Statistically significant correlation was found between aberrant expression of E-cadherin and lymph node metastasis and cell types other than endometrioid adenocarcinoma. Aberrant pattern of gamma-catenin expression was also correlated with deep myometrial invasion. However, alpha-, and beta-catenin expression was not correlated with any clinicopathological parameters. Using the Kaplan-Meier method and log-rank comparison test, abnormal expression of E-cadherin was correlated closely with poor survival (p < 0.05), but cases with loss of both E-cadherin and catenin expression predicted even poorer survival than cases with only one or no aberrant expression in E-cadherin and catenins. We revealed aberrant expression of these cell adhesion molecules among patients with endometrial carcinoma. Aberrant expression of E-cadherin was correlated with lymph node metastasis and cell types other than endometrioid adenocarcinoma, while aberrant expression of gamma-catenin was related with deep myometrial invasion. The expression of E-cadherin might be a possible prognostic factor for endometrial cancer while the expression of catenins may help predict patient's survival.
Adult ; Aged ; Cadherins/*analysis ; Cytoskeletal Proteins/*analysis ; Endometrial Neoplasms/*chemistry/mortality ; Female ; Human ; Immunohistochemistry ; Middle Age ; Trans-Activators/*analysis

Animals ; Glucose ; pharmacology ; Homeodomain Proteins ; analysis ; genetics ; physiology ; Immunohistochemistry ; Insulin ; secretion ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Trans-Activators ; analysis ; genetics ; physiology

OBJECTIVEThis paper studied the effect of RNaseP against CIITA on repressing class II MHC (MHCII) expression.

METHODSIt was constructed that M1-RNA with guide sequences (GS), recognizing the 629 site of CIITA (M1-629-GS), by PCR from pTK117 plasmid, then was cloned into psNAV (psNAV-M1-629-GS). CIITA target gene was obtained from Raji cell by RT-PCR, and then inserted into pGEM-7zf (+) (pGEM-800). psNAV-M1-629-GS and pGEM-800 were transcribed and then mixed up and incubated in vitro. Stable transfectants of hepatocyte with psNAV-M1-629-GS by nanometer were tested for MHCII induction by recombinant human interferon-gamma (IFN-gamma). mRNA abundance of CIITA was measured by RT-PCR.

RESULTSIt showed that M1-629-GS could exclusively cleave pGEM-800 that formed a base pair with the GS. When induced with IFN-gamma, the expression of HLA-DR, -DP, -DQ on psNAV-M1-629-GS+ hepatocyte was (1.01+/-0.51)%, (4.37+/-1.28)%, (1.98+/-0.42)% respectively, was down-modulated 90.65%, 89.11% and 65.32% compared with control, while the mRNA content of CIITA reduced significantly (P<0.01).

CONCLUSIONM1-629-GS could effectively repress MHCII expressing through cleaving CIITA mRNA. These results provided insight into the future application of it as a new nucleic acid drug against the rejection of hepatic transplantation.

Graft Rejection ; prevention & control ; Histocompatibility Antigens Class II ; analysis ; Humans ; Liver Transplantation ; immunology ; Nuclear Proteins ; genetics ; RNA, Messenger ; analysis ; Ribonuclease P ; pharmacology ; Trans-Activators ; genetics

OBJECTIVETo investigate the expression of beta-catenin and its significance in colorectal neoplasms.

METHODSTissue specimens of normal colorectal mucosa, mucosa adjacent to carcinoma, colorectal adenoma and adenocarcinoma were examined for beta-catenin with immunohistochemistry.

RESULTSBeta-catenin was mainly expressed in the cytomembrane of normal mucosa and mucosa adjacent to cancer (the positive rates were 94.6% and 86.5%, respectively) and also in the cytoplasm (the positive rates were 38.7% and 55.0%, respectively), while its expression was negative in the cell nucleus. In adenoma and adenocarcinoma, beta-catenin was mainly expressed in the cytoplasm (the positive rates were 85.1%,and 93.7%, respectively) and partially in the cell nucleus (the positive rates were 12.8% and 23.4%, respectively). Compared with normal mucosa and mucosa adjacent to cancer, the expression of beta- catenin in the cytomembrane of adenoma and adenocarcinoma was significantly lower (P<0.05), while its expression in the cytoplasm and cell nucleus of adenoma and adenocarcinoma was significantly higher (P<0.05). The positive rates of cytoplasm in highly-and moderately differentiated adenocarcinoma were significantly higher than that in poorly-differentiated adenocarcinoma (the positive rates were 100%, 95.5% and 68.8%, respectively). Beta-catenin expression rate in cytoplasm was correlated with Dukes'stages of adenocarcinoma, which was significantly lower in stage A than in stage B/C.

CONCLUSIONThe expression of beta-catenin is significantly correlated with differentiation and Dukes'stages of colorectal carcinoma and it can be used as an indicator for the prognosis of colorectal carcinoma.

Adenocarcinoma ; chemistry ; pathology ; Adenoma ; chemistry ; pathology ; Colorectal Neoplasms ; chemistry ; pathology ; Cytoplasm ; chemistry ; Cytoskeletal Proteins ; analysis ; Humans ; Immunohistochemistry ; Prognosis ; Trans-Activators ; analysis ; beta Catenin

BACKGROUNDHepatitis B virus (HBV) X protein (HBx) and p53 could mutually down-regulate at transcriptional level and HBx could bind with p53 protein within its transactivation domain and inhibit the function of p53 protein. In recent years, effects of arsenic trioxide (As2O3) on the expression of p53 protein have been widely studied, while little is known about the activity of p53 protein. This study was undertaken to delineate the effect of HBV X gene and As2O3 on p53 protein expression (level and activity) in HepG2 cells by small hairpin RNA (shRNA)-mediated RNA interference (RNAi) technique.

METHODSCell line HepG2 and cells with stable expression of HBV X gene (HepG2-X) were treated with 2 micromol/L As2O3, with corresponding untreated cells serving as controls. Cell lysates and nuclear extracts were extracted. Total level and the relative activity of p53 protein were detected by modified enzyme-linked immunosorbent assay (ELISA). HBV X gene sequence-specific shRNA expression vector (pXi-1 and pXi-2) and sequence-unrelated control (pXi-3) were transfected into HepG2-X. Single cell clone with stable expression of shRNA was selected and exposed to propagating culture. The effect of As2O3 on p53 protein expression and activity was re-observed.

RESULTSTotal p53 protein level was up-regulated and its relative activity ratio was enhanced by As2O3 in HepG2 and HepG2-X cells. The total p53 protein level induced by As2O3 was up-regulated by HBV X gene expression, while its relative activity was significantly suppressed. The suppression was removed after HBV X gene expression was repressed by shRNA.

CONCLUSIONSAs2O3 up-regulates p53 protein expression and enhance its activity. HBV X up-regulates As2O3 induced-p53 protein expression while suppresses its activity.

Arsenicals ; pharmacology ; Cell Line, Tumor ; Enzyme-Linked Immunosorbent Assay ; Humans ; Oxides ; pharmacology ; RNA Interference ; Trans-Activators ; genetics ; Tumor Suppressor Protein p53 ; analysis

OBJECTIVETo study the relationship between the abnormal expression of beta-catenin (beta-cat) and the high expressions of cyclin D1 and c-myc and the occurance, proliferation, infiltration, metastasis and prognosis of pancreatic cancer, and to provide rational basis for the clinical diagnosis and treatment.

METHODSImmunohistochemical PicTure trade mark was used to examine the expressions of beta-cat, cyclin D1 and c-myc in 47 cases of the cancerous tissue of pancreas, 12 cases of the pancreatic intraepithelial neoplasia and 10 cases of normal tissue of pancreas, respectively. Pancreatic cancer proliferation cell nuclear antigen (PCNA) was also tested as the index of the extent of proliferation of the pancreatic cancer.

RESULTSbeta-cat was expressed normally in the 10 cases of the normal pancreatic tissue, while cyclin D1 and c-myc were negative. The expression rates of beta-cat, cyclin D1 and c-myc in the tissues of the pancreatic intraepithelial neoplasia and the pancreatic cancer had no significant difference [6/12 and 68.1% (32/47), 6/12 and 74.5% (35/47), 5/12 and 70.2% (33/47) respectively;P values were all more than 0.05]. The abnormal expression rate of beta-cat was significantly correlated to the metastasis of the pancreatic cancer and the one-year survival rate (both P < 0.05), but had no relation with the size, the extent of differentiation, the activity of proliferation, or infiltration of the pancreatic cancer (both P > 0.05). The expression rate of cyclin D1 was correlated with the proliferation of the pancreatic cancer and the extent of differentiation (both P < 0.05), but not with the size, infiltration, metastasis, or one-year survival rate of the pancreatic cancer (both P > 0.05). The expression rate of c-myc was not correlated with the size, the extent of proliferation, infiltration, metastasis, or one-year survival rate (both P > 0.05), but closely with the proliferation activity of the cancerous tissue of pancreas (P < 0.05). The abnormal expression of beta-cat and the high expressions of cyclin D1 and c-myc had a parallel relationship with the pancreatic intraepithelial neoplasia and pancreatic cancer (both P < 0.05, gamma = 1.000, 0.845, 0.437, 0.452).

CONCLUSIONSThe abnormal expression of beta-cat activates cyclin D1 and c-myc, and results in the unchecked proliferation and differentiation, which may play an important role in the genesis of the pancreatic cancer. The abnormal expression of beta-cat is one of the mechanisms for the spread of pancreatic cancer and an index in the molecular biology to determine the metastasis and prognosis of pancreatic cancer.

Adult ; Aged ; Cyclin D1 ; analysis ; Cytoskeletal Proteins ; analysis ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Pancreas ; chemistry ; Pancreatic Neoplasms ; chemistry ; pathology ; Proliferating Cell Nuclear Antigen ; analysis ; Proto-Oncogene Proteins c-myc ; analysis ; Trans-Activators ; analysis ; beta Catenin

OBJECTIVETo investigate the value of using an AMACR/34betaE12/p63 cocktail and double-staining for the diagnosis of small focal protatic carcinoma and precarcinomatous lesions.

METHODSA total of 130 consecutive cases were examined over a 3-month period, including 105 prostate needle biopsy samples, 6 radical prostatectomy specimens and 19 benign prostatic hyperplasia specimens which were excised transurethra or above pubis. 262 paraffin blocks of all the 1030 ones were stained with hematoxylin and eosin and by immunostains for AMACR, 34betaE12, p63, and an antibody cocktail comprising all the three with double-chromogen reaction. The diagnoses were then made according to the immunostaining, HE staining and clinical information.

RESULTSIn the sections stained by the 3-antibody cocktail, blue-black cytoplasmic staining was observed in the epithelial cells of prostatic carcinoma and high-grade prostatic intraepithelial neoplasia (HGPIN) the basal cells of benign glands were stained red. There were no red basal cells around the blue-black glandular epithelium of carcinoma, but discontinuous or consecutive red basal cells were present around the blue-black glandular epithelium of HGPIN. Prostatic carcinoma was found in 214 paraffin blocks (82%), including 31 small focal carcinoma. HGPIN were observed in 64 paraffin blocks (24%), including focal HGPIN and small gland alveolus HGPIN. AAH was found in one block. No benign glands were simultaneously positive for AMACR and negative for basal cell markers.

CONCLUSIONInmmunohistochemistry studies using a 3-antibody cocktail and double staining can improve the detection rate of small focal prostatic carcinoma and HGPIN.

Biomarkers, Tumor ; analysis ; Humans ; Immunohistochemistry ; methods ; Keratins ; analysis ; Male ; Predictive Value of Tests ; Prostatic Intraepithelial Neoplasia ; diagnosis ; metabolism ; Prostatic Neoplasms ; diagnosis ; metabolism ; Racemases and Epimerases ; analysis ; Staining and Labeling ; methods ; Trans-Activators ; analysis ; Transcription Factors ; Tumor Suppressor Proteins ; analysis

OBJECTIVETo investigate the protein and mRNA expression of E-cadherin and beta-catenin in nonsmall cell lung carcinoma (NSCLC) and to find their correlation with histological type, differentiation, metastasis and prognosis.

METHODSHigh sensitive S-P immunohistochemical method and in situ hibridization were used to detect the protein and mRNA expression of E-cadherin and beta-catenin.

RESULTSImmunohistochemistry revealed that among the 101 cases, the positive rates of E-cadherin and beta-catenin were 68.3% and 81.2% respectively. The abnormal expression rates of these two proteins were 61.4% and 64.4% respectively. There was no significant relationship between E-cadherin and beta-catenin staining and histological type of the tumor (P > 0.05). However, there was a statistically significant difference between well and moderately differentiated cells and poorly differentiated cells (P < 0.05). In cases with lymphatic metastasis, the abnormal expression rates of E-cadherin and beta-catenin were significantly higher than those in nonmetastatic cases (P < 0.05). The mean survival time in cases with abnormal E-cadherin and beta-catenin expression were significantly shorter than that in cases with the expression grading (+ +) approximately (+ + +). In situ hybridization showed that in NSCLC, the positive rate of E-cadherin and beta-catenin mRNA was 38.9% and 47.2% respectively. Their concordant rates with (+ +) approximately (+ + +) protein expression were 78.6% and 82.4%, respectively.

CONCLUSIONSThe concordant rate of E-cadherin and beta-catenin mRNA and protein expression was relatively high. They can be used as markers of prognosis of NSCLC in clinical practice.

Adult ; Aged ; Cadherins ; analysis ; genetics ; Carcinoma, Non-Small-Cell Lung ; chemistry ; mortality ; Cytoskeletal Proteins ; analysis ; genetics ; Female ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Lung Neoplasms ; chemistry ; mortality ; Male ; Middle Aged ; Prognosis ; RNA, Messenger ; analysis ; Survival Rate ; Trans-Activators ; analysis ; genetics ; beta Catenin