0.05) compared with control group at 1 h and 3 h; while ~FL 1 in DEX group at 5 h (660.91?72.95) was significant lower (P

AIM: To study the effect of ERK inhibition on the mitochondrial potential change in dexamethasone (DEX)-induced thymocyte apoptosis. METHODS: ERK activity was inhibited by PD098059 (PD), and 4 experimental groups were set: control, PD only, DEX and PD+DEX. Annexin V-FITC/PI double staining flowcytometry was used to detect apoptotic cells at time points of 3 h, 5 h and 7 h. JC-1 staining flowcytometry was adopted to examine mitochondrial membrane potential (△?m) at time points of 3 h, 7 h and 11 h. RESULTS: By stimulation with 1 ?mol/L DEX, the apoptotic rates of mouse thymocytes at 3 h, 5 h and 7 h were (19.63?0.35)%, (41.84?1.67)% and (67.00?2.43)%, respectively, and had significantly difference from control group (4.98?0.39)%, (6.08?0.33)% and (9.31?0.34)% (P0.05). At 3 h, 7 h and 11 h, the rates of low △?m cells were (21.23?1.43)%, (55.34?1.78)% and (70.88?2.87)%, significantly higher than that in control group (P0.05). CONCLUSION: DEX induces mouse thymocyte apoptosis at least partly through ERK pathway, and ERK inhibition has an important biological significance during this process.

BACKGROUNDTrans-arachidonic acids (TAAs), newly discovered markers of nitrative stress and the major products of nitrogen dioxide (NO2(·))-mediated isomerization of arachidonic acid (AA), represent a new mechanism of NO2(·)-induced toxicity. It has been reported that TAAs were generated in oxygen-induced microvascular degeneration model and TAAs were also generated in a diabetic retinopathy (DR) model. In this study, we examined high glucose-induced nitrative stress damage and TAAs levels and explored the possible mechanisms for DR caused by reactive nitrogen species.

METHODSDiabetic rats were induced by intraperitoneal injection of streptozotocin (STZ) at 60 mg/kg. Bovine retinal capillary endothelial cells (BRECs) were selectively cultured and incubated with normal or high glucose. The serum TAAs and AA in diabetic rats were measured by the gas chromatography and mass spectrometry (GC/MS) method. The ratio of peak area of TAAs to AA with selected ion of 79 was estimated by a group t-test. Thrombospondin-1 (TSP-1) in the rat retinas and BRECs extracts were examined by Western blotting. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) protein was examined by Western blotting in BRECs incubated with high glucose.

RESULTSThe TAAs to AA ratio (TAAs/AA) was significantly increased in the serum at 8, 12 and 16 weeks after STZ injection (P < 0.05), with no noticeable change found at 2 or 4 weeks (P > 0.05). Expression of TSP-1 in the retina of diabetic rats was progressively elevated according to the duration of diabetes. TSP-1 expression was increased in BRECs incubated with high glucose at 48 hours. Moreover, high glucose also increased ERK1/2 expression, which peaked at 30 minutes and then decreased in the following 48 hours.

CONCLUSIONAn elevation of TAAs/AA is associated with high glucose-induced nitrative stress, which probably involves upregulation of TSP-1 through activating ERK1/2.

Animals ; Arachidonic Acid ; metabolism ; Blotting, Western ; Cattle ; Cells, Cultured ; Diabetes Mellitus, Experimental ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Gas Chromatography-Mass Spectrometry ; Male ; Rats ; Rats, Sprague-Dawley ; Reactive Nitrogen Species ; metabolism ; Streptozocin ; Thrombospondin 1 ; genetics ; Up-Regulation

ONYX-015 and H101 are E1B 55-kDa protein-deficient replicating C group adenoviruses that are currently in clinical trials as antitumor agents. However, their application in cancer gene therapy is limited by the native tropism of C group adenoviruses. This is in part due to low expression of the C group adenovirus receptor (coxsackievirus-adenovirus receptor, CAR) on malignant tumors. An H101-based chimeric virus vector containing sequences encoding the Ad35 fiber domain instead of the Ad5 fiber (H101-F35) was constructed. This modification allowed infection of tumor cells through CD46, a membrane protein over-expressed on tumors. The CAR and CD46 RNA expression was evaluated by RT-PCR method. H101-F35 conferred a stronger cytocidal effect than H101 and ONYX-015 in tumor cell lines that lacked CAR expression (MDA-MB-435 and MCF-7), while the cytocidal effect of H101-35, H101 and ONYX-015 was similar in high-level CAR expressing cancer cell lines (A549, NCI-H446, Hep3B, LNCaP, ZR-75-30 and Bcap-37). In an MDA-MB-435 xenograft mouse tumor model, tumor growth in mice receiving H101-F35 was significantly inhibited compared with mice injected with H101. These results suggest that the chimeric oncolytic adenovirus H101-F35 vector might be a useful candidate for gene therapy of cancer.

OBJECTIVETo investigate the diagnostic applications of endometrial intraepithelial neoplasia (EIN), and the expression of PTEN in endometrial lesions.

METHODSFifty-one cases of endometrial lesions were enrolled in this study. Using diagnostic criteria of EIN, the diagnosis were made and compared with the original results. Immunohistochemistry for PTEN was performed in all cases.

RESULTSTwo cases of simple hyperplasia originally diagnosed were reclassified as EIN. Three cases with atypia originally diagnosed showed no EIN pattern. PTEN deletion rates were 50.0%, 50.0%, 66.7% and 81.8% in proliferative endometrium, benign hyperplasia, EIN and endometrial carcinoma, respectively.

CONCLUSIONSDiagnosis of EIN is applicable and its morphology and diagnostic criteria are different from the classical one (WHO94) for endometrial hyperplasia. Detection of PTEN deletion by immunohistochemistry is useful in identifying EIN, but cannot be used as an ultimate confirming factor.

Adult ; Aged ; Carcinoma, Endometrioid ; metabolism ; pathology ; Endometrial Hyperplasia ; metabolism ; pathology ; Endometrial Neoplasms ; metabolism ; pathology ; Female ; Humans ; Middle Aged ; PTEN Phosphohydrolase ; metabolism ; Precancerous Conditions ; metabolism ; pathology ; Young Adult

OBJECTIVETo deduce the protocol, scoring criteria and interpretive guidelines for assessment of HER2 gene expression status by fluorescence in-situ hybridization (FISH) and to compare the results with those obtained by immunohistochemistry.

METHODSThe HercepTest kit from Dako Cytomation was employed for immunohistochemistry. FISH for HER2 gene expression status was performed using PathVysion DNA probe kit on the archival paraffin-embedded sections of breast cancer tissues from 28 Chinese female patients with immunohistochemical staining scores of (3 +), (2 +), (1 +) and 0.

RESULTSTen of the 12 patients with score (3 +) by immunohistochemistry were positive for HER2 by FISH, with 2 cases being polysomy. Two other cases with FISH-negative were also shown to be polysomy. Seven of the 10 patients with score (2 +) by immunohistochemistry showed HER2 gene amplification, with 1 case being polysomy. Two of the remaining 3 cases, which were FISH-negative, were shown to be polysomy. All the patients with scores (1 +, number = 3 ) or 0 ( number = 3) by immunohistochemistry failed to show amplification. One case of polysomy was noted in either group.

CONCLUSIONSImmunohistochemistry is useful as an initial screening tool for HER2 expression status. Because of the obvious discrepancies between protein expression and gene amplification, patients with score (2 +) by immunohistochemistry should undergo FISH testing as well. FISH is also required in selected examples with score (3 +) immunohistochemical results, especially in those with false-positive immunohistochemistry due to chromosome 17 aneuploidy.

Breast Neoplasms ; genetics ; metabolism ; Carcinoma, Ductal, Breast ; genetics ; metabolism ; Chromosomes, Human, Pair 17 ; Female ; Gene Amplification ; Gene Expression Regulation, Neoplastic ; Genes, erbB-2 ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Polyploidy ; Receptor, ErbB-2 ; metabolism

OBJECTIVETo study the effect of p38 on the cycloheximide (CHX)-induced HL-60 cell death through mitochondria pathway.

METHODSInhibition of p38 pathway was by SB203580 (SB). Four groups were set up: control, SB only, CHX only and SB + CHX. Sub-diploid cell ratio was detected by PI staining flow cytometry at 6, 9, 12, 18, 24 h time points, and apoptotic cell ratio by Annexin V-FITC/PI double staining flow cytometry at 6 h and 18 h time points. High J-aggregate cells were evaluated by the J-aggregate contents, measurement of the J-aggregate (FL2) and J-monomer (FL1) by JC-1 flow cytometry, calculation of the delta psi m by FL2/FL1 and analysis of the delta psi m changes at 18 h time points.

RESULTSThe sub-diploid cell ratio in CHX group was significantly higher than that in control group at 6 h time point, and the ratio in SB + CHX group was significantly higher than that in CHX group at 9 h time point. At 18 h time point the apoptotic cell ratios in both CHX and SB + CHX groups were significantly higher than those in control group (P < 0.01). There was no significant difference of apoptotic cell ratio between CHX group and SB + CHX group (P > 0.05). At 18 h time point the necrotic cell ratios in both CHX and SB + CHX groups were significantly higher than that in control group (P < 0.01); and that in SB + CHX group was significantly higher than that in CHX group (P < 0.01). The high J-aggregate cell ratios in CHX and SB + CHX groups were significantly lower than that in control group (P < 0.05), and that was signficantly lower in SB + CHX group than in CHX group (P < 0.01). For the FL2/FL1 value (delta psi m) CHX group (0.17 +/- 0.01) and SB + CHX group (0.05 +/- 0.003) were significantly higher than control group (0.38 +/- 0.02) (P < 0.01), and SB + CHX group was significantly lower than CHX group (P < 0.01).

CONCLUSIONCHX can induce HL-60 cell apoptosis and the cell mitochondria depolarization, and the latter was intensified by inhibition of the p38 pathway. p38 pathway may related to the cell necrosis in the cycloheximide-induced HL-60 cell apoptosis model. s

Apoptosis ; drug effects ; Cycloheximide ; pharmacology ; HL-60 Cells ; Humans ; Membrane Potentials ; Mitochondria ; physiology ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

BACKGROUNDPancreatic cancer is one of the most common tumors and has a 5-year survival for all stages of less than 5%. Most patients with pancreatic cancer are diagnosed at an advanced stage and therefore are not candidates for surgical resection. In recent years, investigation into alternative treatment strategies for this aggressive disease has led to advances in the field of gene therapy for pancreatic cancer. E. coli purine nucleoside phosphorylase/6-methylpurine deoxyribose (ePNP/MePdR) is a suicide gene/prodrug system where PNP enzyme cleaves nontoxic MePdR into cytotoxic membrane-permeable compounds 6-methylpurine (MeP) with high bystander activity. hTERT is expressed in cell lines and tissues for telomerase activity. In this study we examined the efficacy of ePNP under the control of hTERT promoter sequences and assessed the selective killing effects of the ePNP/prodrug MePdR system on pancreatic tumors.

METHODSRecombinant pET-PNP was established. The protein of E. coli PNPase was expressed and an antibody to E. coli PNPase was prepared. Transcriptional activities of hTERT promoter sequences were analyzed using a luciferase reporter gene. A recombinant phTERT-ePNP vector was constructed. The ePNP/MePdR system affects SW1990 human pancreatic cancer cell lines in vitro.

RESULTSThe hTERT promoter had high transcriptional activity and conferred specificity on cancer cell lines. The antibody to E. coli PNPase was demonstrated to be specific for the ePNP protein. The MePdR treatment induced a high in vitro cytotoxicity on the sole hTERT-ePNP-producing cell lines and affected SW1990 cells in a dose-dependent manner.

CONCLUSIONSThe hTERT promoter control of the ePNP/MePdR system can provide a beneficial anti-tumor treatment in pancreatic cancer cell lines including a good bystander killing effect.

Cell Line, Tumor ; Escherichia coli ; enzymology ; Genetic Therapy ; Humans ; Pancreatic Neoplasms ; therapy ; Promoter Regions, Genetic ; Purine Nucleosides ; therapeutic use ; Purine-Nucleoside Phosphorylase ; genetics ; Telomerase ; genetics

OBJECTIVETo explore epidermal growth factor receptor (EGFR) and HER2 gene status, to assess the correlation between EGFR and HER2 gene status, and to investigate the role of copy number increase and amplification of EGFR gene and HER2 gene in the tumorigenesis and disease progression of non-small-cell lung cancer.

METHODSUsing Path Vysion kit and LSI EGFR SpectrumOrange/CEP7 Spectrum Green probes, EGFR gene and HER2 gene status were evaluated by fluorescence insitu hybridization (FISH) using formalin-fixed, paraffin-embedded samples from 31 patients with non-small-cell lung cancer, including 20 adenocarcinomas, 2 squamous cell carcinomas, 2 large cell carcinoma, 4 bronchoalveolar carcinomas and 3 adenosquamous carcinomas. The correlation between EGFR and HER2 gene status was analyzed.

RESULTSSix of thirty-one carcinomas showed EGFR gene amplification. Of 25 cases without EGFR gene non-amplification, four tetrasomy and 5 polysomy were detected. Overall, 15 out of 31 carcinomas demonstrated either EGFR gene copy number increase or gene amplification (15/31). HER2 gene amplification was seen in 2 of the 31 cases. Four trisomy, one tetrasomy and nine polysomy cases were found in 29 tumors that had no HER2 gene amplification. Overall, 16 of 31 cases showed either HER2 gene copy number increase and/or amplification (16/31). Synchronous EGFR and HER2 gene numerical changes, i.e. gene copy number increase and gene amplification, were found in 12 of 31 cases (12/31), and almost all such patients had either clinical stage III or IV tumor. EGFR gene numerical changes significantly correlated with HER2 gene abnormality (chi(2)(Adj) = 7.3045, P = 0.0069).

CONCLUSIONSEGFR or HER2 copy number increase is much more frequent than gene amplification in no-small-cell lung cancer. Our data based on gene alterations indicate, for the first time, that there is a significant correlation between EGFR alterations and HER2 abnormalities. Both genes are involved in the tumorigenesis and development of lung cancer. EGFR/HER2 dimer is one of the predominant heterodimerization types in lung cancer. The interactions between EGFR and HER2 may play a rule in the progression of non-small-cell lung cancer.

Carcinoma, Non-Small-Cell Lung ; genetics ; pathology ; Chromosomes, Human, Pair 17 ; Chromosomes, Human, Pair 7 ; Gene Amplification ; Gene Dosage ; Gene Expression Regulation, Neoplastic ; Genes, erbB-1 ; genetics ; Genes, erbB-2 ; genetics ; Humans ; In Situ Hybridization, Fluorescence ; Lung Neoplasms ; genetics ; pathology ; Polyploidy

OBJECTIVETo study the HER2 gene status (by fluorescence in situ hybridization (FISH) in breast cancer with HER2 protein overexpression, the correlation between gene amplification and protein overexpression, as well as the rate and significance of chromosome 17 aneusomy.

METHODSOne hundred and twenty archival cases of breast cancer with formalin-fixed and paraffin-embedded tumor tissues with 2+ (42 cases) and 3+ (78 cases) HER2 protein overexpression by immunohistochemistry (IHC, HercepTest, Dako) were tested by FISH (PathVysion, Vysis) for HER2 gene status. The rate of chromosome 17 aneusomy was also analyzed.

RESULTSAmongst the 42 samples with IHC 2+, HER2 gene amplification was identified in 32 cases (76.19%), which included 11 cases with low amplification (ratio 2 approximately 4), 20 cases with moderate amplification (ratio 4 approximately 10) and 1 case with high amplification (ratio>10). Amongst the 78 samples with IHC 3+, HER2 gene amplification was identified in 71 cases (91.03%), which included 9 cases with low amplification, 48 cases with moderate amplification and 14 cases with high amplification. Chromosome 17 aneusomy was found in 83 cases (83/120, 69.17%), in which 14 cases (11.67%) showed hypodisomy (chromosome 17 copy number per cellor=3.76). Amongst the 42 cases with IHC 2+, 25 samples (59.52%) had chromosome 17 aneusomy, including 3 cases with hypodisomy, 18 cases with low polysomy and 4 cases with high polysomy. Amongst the 78 cases with IHC 3+, 58 samples (74.36%) had aneusomy 17, including 11 cases with hypodisomy, 34 cases with low polysomy and 13 cases with high polysomy. Most of IHC 2+ and FISH-positive cases had low or moderate HER2 gene amplification, while most of the IHC 3+ and FISH-positive cases had moderate or high gene amplification (P=0.0003). Six of the 7 samples with IHC 3+ and FISH-negativity had chromosome 17 aneusomy and 5 of the 10 samples with IHC 2+ and FISH-negativity had such aneusomy.

CONCLUSIONSA high concordance rate is noted between IHC 3+ and FISH positive results. The rate of FISH positive in IHC 2+ patients was higher than reported in other studies. Low or moderate HER2 gene amplification in IHC 2+ and moderate or high gene amplification in IHC 3+ occurs quite frequently. Chromosome 17 aneusomy (including hypodisomy, low polysomy and high polysomy) is also a relatively common phenomenon in our cohort with HER2 overexpression, with predominance of low polysomy.

Adult ; Aged ; Aged, 80 and over ; Aneuploidy ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; genetics ; metabolism ; pathology ; Chromosomes, Human, Pair 17 ; genetics ; Female ; Gene Amplification ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Middle Aged ; Receptor, ErbB-2 ; genetics ; metabolism ; Young Adult