OBJECTIVETo investigate the expression of cytokeratins and ret in thyroid papillary carcinoma (TPC) and their diagnostic value.

METHODSDuring the period of October 1999 to March 2002, 69 cases of TPC (42 cases with adjacent normal thyroid tissue) and 14 cases of nodular goiter with papillary hyperplasia were enrolled into the study. Immunohistochemistry for CK19, CK17, CK8, CK20 and ret was performed in all cases using EnVision and LSAB methods respectively.

RESULTSThe positive rates for CK19 and ret in TPCs were 85.5% and 68.1% respectively, which were significantly (P < 0.01) higher than those in nodular goiter and normal thyroid tissue (25.0% and 5.4% respectively). The expression of CK17 was also observed in a few cases of TPCs (11/69, 15.9%), which was mainly localized in areas of squamous metaplasia, poorly differentiated carcinoma and/or in the small infiltrative foci. The positive rates for CK8 were 75.4% and 26.8% in TPCs and benign thyroid tissue respectively. All cases were negative for CK20.

CONCLUSIONSCK19, CK17 and ret expressions are significantly higher in TPCs than benign thyroid tissue; and this characteristic can have important diagnostic value.

Carcinoma, Papillary ; metabolism ; pathology ; Humans ; Immunohistochemistry ; Keratins ; analysis ; biosynthesis ; Proto-Oncogene Proteins ; biosynthesis ; Proto-Oncogene Proteins c-ret ; Receptor Protein-Tyrosine Kinases ; biosynthesis ; Thyroid Neoplasms ; metabolism ; pathology

The aim of study was to investigate the killing effect of double suicide gene system mediated by retroviral vector on K562 cells in vivo and ex vivo. CDglyTK gene was transfected into PA317 cells by using lipofectamine. K562 cells were infected with viral supernatant. K562/CDglyTK cells were treated with 5-fluorocytosine (5-FC) and/or ganciclovir (GCV). Mice were randomly divided into three groups: tumor formation, tumor inhibition and tumor therapy. Each mouse was implanted with K562/CDglyTK cells or K562 cells. The results indicated that the killing effect of 5-FC in combination with GCV on K562/CDglyTK was more significant than using 5-FC or GCV alone. In vivo study showed that after being injected subcutaneously with K562 cells and K562/CDglyTK cells, there was not obvious difference in tumor formation rate of mice, 5-FC + GCV could suppress tumor formation of the K562/CDglyTK cells. After being treated with 5-FC and GCV, the median tumor volume of mice implanted with K562/CDglyTK cells decreased obviously, compared with the control group. Their median survival was significantly prolonged. It is concluded that double suicide genes are more effective for killing effect on K562 cells in vivo and in ex vivo. It may be applicable to clinical gene therapy.
Cytosine Deaminase ; genetics ; Flucytosine ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; K562 Cells ; Protein-Tyrosine Kinases ; genetics ; Receptor Protein-Tyrosine Kinases ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; genetics ; Recombination, Genetic ; Retroviridae ; genetics

OBJECTIVESTo investigate the effects of Arg-Gly-Asp-Ser (RGDS) tetrapeptide on integrin signaling and apoptosis in fibronectin (FN) -stimulated hepatic stellate cells (HSCs).

METHODS3H-thymidine incorporation, annexin-V/propidium iodide double-labeled flow cytometry (FCM) and transmission electron microscopy were employed to estimate the influence of RGDS on the proliferation and apoptosis of HSCs. And the adhesion rates were observed by toluidine blue colorimetric assay. The expression of focal adhesion kinase (FAK) mRNA and protein in HSCs was detected using RT-PCR and western blotting analysis, respectively.

RESULTSRGDS tetrapeptide at the concentrations of 25 microg/ml, 50 microg/ml and 100 microg/ml inhibited the proliferation of HSCs and induced HSCs apoptosis in dose-dependent and time-dependent manners, with the apoptotic rates of 9.49%, 27.67%, 31.59%, and the necrotic rates of 3.47%, 5.38%, 9.10%, respectively. Both the rates were higher than those in FN group (apoptotic rate: 3.44%; necrotic rate: 2.39%), F=8.02, P<0.05. After adding RGDS tetrapeptide to HSCs for 2 hours, the adhesive inhibition rates were 8.82%, 29.41% and 45.59%, respectively, which were higher than that in FN group (F=20.58, P<0.01). After exposure of HSCs to RGDS tetrapeptide for 24 hours, FAK protein decreased, and FAK mRNA was down-regulated earlier, about 2 hours after exposure to RGDS tetrapeptide.

CONCLUSIONThese results suggest that RGDS tetrapeptide may induce apoptosis of HSC in both dose-dependent and time-dependent manners in vitro, which may be related to the disruption of cell matrix adhesion and down-regulation of FAK expression.

Apoptosis ; drug effects ; Fibronectins ; pharmacology ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Hepatocytes ; cytology ; physiology ; Humans ; Integrins ; metabolism ; physiology ; Liver ; cytology ; physiology ; Oligopeptides ; pharmacology ; Platelet Aggregation Inhibitors ; pharmacology ; Protein-Tyrosine Kinases ; biosynthesis ; genetics ; metabolism ; Signal Transduction

Novel Oncogene with Kinase-domain (NOK) is a novel tumor-related gene, coding receptor like protein with a kinase domain. Overexpression of NOK leads to tumorigenesis and metastasis. To further study NOK function in physiological condition, it is necessary to prepare the anti-NOK antibody. In this report, GST fusion protein was adopted to prepare polyclonal antibodies against hNOK. The result showed that the antibodies we generated is with a very high titriation, and can be used for examination of NOK protein by Westernblot. Furthermore, the antibodies were used for immunohistochemistry in lung cancer tissues, and the results demonstrated high expression of hNOK in the tumor tissues. The antibody of hNOK we generated can serve as a diagnostic method for the lung cancer.
Animals ; Antibodies ; genetics ; metabolism ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Biomarkers, Tumor ; genetics ; metabolism ; Humans ; Lung Neoplasms ; diagnosis ; genetics ; metabolism ; Mice ; Oncogene Proteins ; biosynthesis ; genetics ; immunology ; RNA, Messenger ; biosynthesis ; genetics ; Receptor Protein-Tyrosine Kinases ; biosynthesis ; genetics ; immunology

OBJECTIVETo investigate the effect and mechanisms of tumor suppressor gene PTEN on the induction of anoikis of hepatocellular carcinoma SMMC-7721 cells.

METHODSSMMC-7721 cells were transfected with GFP plasmids containing wild-type PTEN or phosphatase inactivating mutant PTEN (C124A-PTEN) in vitro; The PTEN expression and the phosphorylation levels of focal adhesion kinase (FAK) and protein kinase B (PKB/Akt) were detected by Western blotting; Flow cytometry assay and laser scanning confocal microscopy were used to analyze apoptosis in adherent and non-adherent cells.

RESULTSCompared with the control, PTEN expression in the cells transfected with wild-type PTEN increased to 248%, while the phosphorylation level of FAK and Akt decreased 65.2% and 89.1%, respectively; and the anoikis percentage increased from 9.5% to 31.3%. In the cells transfected with C124A-PTEN, neither the phosphorylation of FAK and Akt nor the anoikis percentage had obviously changed, although the PTEN expression enhanced dramatically in comparison with the control.

CONCLUSIONThrough its phosphatase activity, tumor suppressor gene PTEN can suppress the phosphorylation of FAK and Akt, and induce anoikis in hepatocellular carcinoma cells.

Anoikis ; physiology ; Carcinoma, Hepatocellular ; pathology ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; Humans ; Liver Neoplasms ; pathology ; PTEN Phosphohydrolase ; biosynthesis ; genetics ; Phosphoric Monoester Hydrolases ; metabolism ; Phosphorylation ; Tumor Cells, Cultured

OBJECTIVETo study the role of focal adhesion kinase (FAK) in the pathogenesis of human hypertrophic scar.

METHODSHuman hypertrophic scar fibroblasts (HSFB) were isolated from human hypertrophic scar and cultured in vitro. The cells were then divided into 3 groups as AT group (phosphorothioate FAK ASODN was transfected into the HSFB by liposome), LPC group (liposome only), and LC group (control group, without liposome or ASODN). The FAKmRNA index of HSFB was assessed by polymerase chain reaction method (FQ-PCR). The collagen synthesis of HSFB was assessed by 3H-proline incorporation method.

RESULTSThe FAK mRNA index of HSFB in AT group 48 hours after transfection was significantly lower than that in LPC and LC groups (0.043 +/- 0.030, 0.124 +/- 0.070, 0.127 +/- 0.0195, P < 0.05). The 3H-proline incorporation rate in AT group was lower than that in LPC and LC groups (257.0 +/- 15.14, 962.2 +/- 300.5, 930.8 +/- 28.97, P < 0.01).

CONCLUSIONThe expression of FAK gene and collagen synthesis of the cultured HSFB could be inhibited by FAK ASODN, indicating that FAK played a role in the development of excessive fibrosis of human hypertrophic scar.

Cells, Cultured ; Cicatrix, Hypertrophic ; genetics ; metabolism ; pathology ; Collagen ; biosynthesis ; Fibroblasts ; drug effects ; metabolism ; Focal Adhesion Protein-Tyrosine Kinases ; genetics ; metabolism ; Humans ; Oligonucleotides, Antisense ; genetics ; pharmacology ; Transfection

The purpose of this study was to detect the serum PTK7 level of patients with acute lymphocytic leukemia, and to reveal its clinical value for diagnosis of diseases. A total of 136 patients diagnosed as acute lymphocytic leukemia from May 2012 to April 2014 in our hospital were enroled in this study and were divided into the L1 group (n = 42), L2 (n = 45) and L3 group (n = 49) according cytomorphology, and 48 normal children were selected as control group. Fluorescence quantitative PCR was used to detect mRNA level of PTK7 in peripheral blood mononuclear cells, and Western blot was used to detect PTK7 protein expression. The results showed that the PTK7 mRNA level in L1 group was significantly higher than that in normal group (P = 0.000) . The PTK7 mRNA level in L2 group was significantly higher than that in the L1 group (P = 0.000). The PTK7 mRNA level in L3 group and L2 group had not significantly different between each other (P = 0.123). Serum PTK7 protein level in L1 group was very significantly higher than that in normal group (P = 0.000) . The serum PTK7 protein level in L2 group were very significantly higher than that in the L1 group (P = 0.003) and serum PTK7 protein level in L3 and L2 group had no significance difference (P = 0.312) . It is concluded that the expression level of serum PTK7 protein has a potential clinical value for the diagnosis of acute lymphocytic leukemia, but without specificity for ALL subsets.
Cell Adhesion Molecules ; blood ; genetics ; Humans ; Leukocytes, Mononuclear ; metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; blood ; diagnosis ; genetics ; RNA, Messenger ; biosynthesis ; blood ; genetics ; Receptor Protein-Tyrosine Kinases ; blood ; genetics

Animals ; Carcinoma, Papillary ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Gene Rearrangement ; Humans ; Oncogene Proteins ; biosynthesis ; genetics ; Oncogene Proteins, Fusion ; Prognosis ; Protein-Tyrosine Kinases ; Thyroid Neoplasms ; genetics ; metabolism

To study the effects of tyrosine-kinase inhibitor STI571 on the adhesion of RPMI8226 cells to fibronectin (FN), cell adhesion mediated adriamycin-resistance and the Rac1 mRNA expression, the adhesion of RPMI8226 cells to fibronectin and drug resistance mediated by cell adhesion were determined by means of crystal violet staining and MTT assays respectively, Rac1 mRNA levels in RPMI8226 cells were examined by semi-quantitative RT-PCR. The results showed that STI571 could inhibit the adhesion of RPMI8226 cells to fibronectin. When RPMI8226 cells had been adhered to FN or BSA-coated wells for 1, 6 and 12 hours, the adhesion rates were (43.71 +/- 2.18)%, (55.63 +/- 1.56)%, and (63.42 +/- 2.46)% respectively. After treatment with STI571 20 micromol/L, the adhesion rates decreased to (15.12 +/- 1.04)%, (17.58 +/- 1.32)% and (17.24 +/- 1.59)% respectively (P < 0.05). The experiment revealed that growth of RPMI8226 cells adhered to FN-coated plates had a significant advantage over growth on BSA-coated plates when exposed to adriamycin (Adr) for 1 hour followed by a 24-hour culture period, and the mean IC(50) value for FN-adhered cells was (1.46 +/- 0.04) micromol/L while mean IC(50) value for BSA control was (0.78 +/- 0.03) micromol/L (P < 0.05). Following treatment with 20 micromol/L STI571, the mean IC50 values for FN and BSA adhered cells were (0.81 +/- 0.05) micromol/L, (0.74 +/- 0.02) micromol/L respectively, there was no significant difference between them (P > 0.05). RT-PCR demonstrated that the relative Rac1 mRNA level (Rac1/GAPDH) in RPMI8226 cells was downregulated following being treated with 20 micromol/L STI571. It is concluded that STI571 can inhibit the adhesion of RPMI8226 cells to fibronectin, reverse cell adhesion mediated adriamycin-resistance, and downregulate Rac1 mRNA level.
Benzamides ; Cell Adhesion ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Fibronectins ; metabolism ; Humans ; Imatinib Mesylate ; Multiple Myeloma ; metabolism ; pathology ; Piperazines ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Pyrimidines ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Tumor Cells, Cultured ; rac1 GTP-Binding Protein ; biosynthesis ; genetics

AIMTo study the effects of FAK-ERK1/2 signaling pathway and FAK antisense oligodeoxynucleotides (ODNs) on vascular smooth muscle cell (SMC) migration and adhesion stimulated by fibronectin (FN).

METHODSMigration and adhesion of cultured SMCs were stimulated by different concentrations of FN, FAK, ERK1/2. And their phosphorylation were detected by immunoprecipitation and Western blot. FAK antisense ODNs were transfected into SMCs by cationic lipid to investigate its modulatory effects on tyrosine phosphorylation, SMCs migration and adhesion were also measured by modifing Boyden Chamber and morphological enumeration, respectively.

RESULTSFAK were expressed when SMCs adhesion and migration were successfully simulated by FN (5, 10, 20, 40, 60 micrograms.mL-1), high contents of FAK and ERK1/2 phosphorylation were detected by 20 micrograms.mL-1 FN or more. FAK antisense ODNs were transfected efficiently by cationic lipid. FAK and ERK1/2 phosphorylation were inhibited magnificently after FAK antisense ODNs transfection. Cell migration stimulated by FN 10, 20, 40 and 60 micrograms.mL-1 were reduced by 23.26%, 21.63%, 19.31% and 17.88% respectively (P < 0.05). SMCs adhesive spreading in 5-60 micrograms.mL-1 FN groups were reduced by 17.89%-27.67% (P < 0.05).

CONCLUSIONFAK-ERK1/2 mediated signal transduction play important roles in SMCs migration and adhesion stimulated by extracellular matrix. The process can be inhibited by FAK antisense ODNs effectively.

Animals ; Aorta ; cytology ; Cell Adhesion ; drug effects ; Cell Movement ; drug effects ; Cells, Cultured ; Fibronectins ; pharmacology ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Mitogen-Activated Protein Kinase 1 ; biosynthesis ; metabolism ; Mitogen-Activated Protein Kinase 3 ; Mitogen-Activated Protein Kinases ; biosynthesis ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; genetics ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Transfection