Objective To study the inhibition of lung cancer cell line by telomerase anti\|sense DNA,and discuss the possibility of using it in clinical treatment. Methods A phosphorothioate oligonucleotide(PS\|ODN) with sequence identical to the repeat sequence of the mammalian telomere 5′\|d(TTAGGG)\|3′ and a control scrambled sequence 5′\|d(TGTGAG)\|3′ were incubated with a lung cancer cell line.The effects of PS\|ODN on cell line growth,colony\|forming and growth shape were detected.The in vivo efficacy of this PS\|ODN was evaluated in a 801\|D nude mouse model.Once tumors were established these animals were administered PS\|ODN or saline for 15 days. Results Telomease anti\|sense DNA inhibit telomerase activation of cell line 801\|D growth and colony\|forming.The activity of the 6\|mer telomere mimic demonstrated a dose dependency.No activity was observed with the scrambled controls.A significant decrease in tumor weight was observed in animals given PS\|ODN, but not followig saline\|treated animale.Conclusion\ These results demonstrated that short hexameric oligonucleotide telomere exerts the growth inhibitory effect on lung cancer cell in vitro and in vivo, and suggest the potential utility of telomerase anti\|sense DNA as cancer cell inhibitors.\;[

BACKGROUNDThe bovine herpersvirus structural protein BVP22 exhibits the remarkable property of intercellular trafficking whereby the protein fused to BVP22 spreads from the cell in which it is synthesized to surrounding cells. This function of BVP22 might be exploited to overcome the low efficiency of genes and gene products delivery, which is a major hurdle in gene therapy. The aim of this study is to investigate the cellular localization and intercellular trafficking of BVP22 in vitro and in vivo and provide scientific data for its application in gene therapy of human lung cancer.

METHODS801D cells were transfected respectively with plasmids pEYFP and pEYFP-BVP22 mediated by Lipofectamin and were selected by G418 to establish clone cell lines. The expression and cellular localization of BVP22 were examined by direct observation of YFP. Intercellular trafficking of BVP22 in vitro was detected by fluorescence-activated cell sorting (FACS) and immunocytochemical staining with YFP-antibody. Subcutaneous 801D tumors in nude mice were established to investigate the intercellular trafficking of BVP22 in vivo.

RESULTSClone cell lines pEYFP-801D and pEYFP-BVP22-801D were established successfully. Cellular localization of BVP22 displayed heterogenic- ity. BVP22 was present in nuclei in most cells and only a few cells showed filamentous cytoplasm pattern. The results of FACS showed that the ratios of YFP-positive cells in mixed cells did not enhanced significantly. Immunocytochemical staining demonstrated that the nuclei of almost all cells were stained positively after pEYFP-BVP22-801D and 801D were cultured for 24h. Intercellular trafficking of YFP-BVP22 could be observed in subcutaneous 801D tumors in nude mice by immunohistochemical staining.

CONCLUSIONSBVP22 displays nuclear localization in most cells. Its nuclear localization might be related to cell mitosis and intercellular trafficking. BVP22 can mediate intercellular trafficking of fusion protein in vitro and in vivo.

BACKGROUNDWith the development of antibody technology, more and more immunoconjugates are used in clinical treatment for different cancers. The aim of this study is to investigate the inhibitive effects of 5F11-DOX immunoconjugate on human lung adenocarcinoma cell line LTEP-A2 in vitro and in vivo and to explore the potential mechanism.

METHODSThe 5F11-DOX immunoconjugate was produced by diluted glutaraldehyde crosslinking. The killing efficiency of 5F11-DOX was detected by clonogenic assay. The distribution of DOX was observed under fluorescence microscope and the 5F11 location was determined by immunohistochemistry. The therapeutic efficacy of 5F11-DOX and free DOX was detected on subcutaneous or intraperitoneal exnogenic transplanted tumors of human lung adenocarcinoma A2 cells in nude mice.

RESULTS5F11-DOX of 0.04mg/L could kill all the A2 cells in vitro and the killing efficiency was 10 times as that of the free DOX. Fluorescence microscopy showed that fluorescence of DOX in 3mg/L 5F11-DOX group was much stronger than that in 3mg/L free DOX group after treating A2 cells with 3mg/L 5F11-DOX or DOX for 3h, then incubating the cells with fresh medium for another 24 hours. Immunohistochemistry showed that 5F11 located in cell membrane and cytoplasm and fluorescence microscopy proved that DOX located inside the cells. The average sizes of subcutaneous or intraperitoneal exnogenic transplanted tumors in 5F11-DOX group were obviously smaller than those of the control group and free DOX group at the same dosage (P < 0.05), and the anti-tumorogenicity efficacy of 5F11-DOX was 4-8 times as that of free DOX. The HE staining showed that extensive necrosis occurred in the center of tumors and around cancer nests in 5F11-DOX group.

CONCLUSIONSThe killing efficacy of 5F11-DOX on human lung adenocarcinoma cell line A2 is obviously higher than that of the free DOX.

Objective　To evaluate the inhibition effect of immunoconjugate of doxorubicin(DXR) with a monoclonal antibody, 5F11 on human lung cancer cells and its reversal effect on resistant lung cancer cells to chemotherapeutic drug. Methods　DXR was attached to 5F11 using dilute glutaraldehyde crossing.The antitumor activity of immunoconjugate, 5F11-DXR, against the sensitive antigen-positive cell line, A2, drug-resistant antigen-positive cell lines, 801-D and 801-DDXR, and antigen-negative cell line, ascite cancer cell was evaluated by human tumor cell cloning assay and dye exclusion assay. Results　According to the results of various assays, comparing with single DXR, 5F11-DXR could significantly increase the cytotoxicity to A2, 801-D and 801-DDXR cell lines with a DXR concentration of 0.4*!μg／ml(P＜0.05), and this difference was even more distinct to A2 cell line with lower concentration of DXR (0.04*!μg/ml). However, there was no remarkable difference between 5F11-DXR and single DXR in cytotoxicity to antigen-negative ascite cancer cell(P＞0.05). Conclusion　5F11-DXR can remarkably increase the cytotoxicity of DXR to the sensitive target cells and even effectively reverse the drug-resistant cell lines to DXR. There is no significant difference between 5F11-DXR and DXR in killing antigen-negative cancer cells.

BACKGROUNDTo isolate and clone the cisplatin genes in 801-D cell line, a kind of lung cancer cell line, with the emphasis of the objective genes regulated by wild type p53 (wtp53).

METHODSTotal RNA was extracted from transfected 801-D-wtp53, 801-D-vector cells which were both treated by cisplatin and 801-D-wtp53 cells. Using mRNA differential display, the DNA bands on gel were displayed by silver stain method. The DNA bands obtained from differential display were recovered and reamplified by PCR. The isolated genes were further proved by reverse Northern dot blot and were cloned to pGEMT easy vector.

RESULTSSix positive genes were identified and cloned. Out of them, 2 related fragments were found to have an open reading frame. One was partly homologous to ribonucleoside-diphosphate reductase A, and the other was no homologous to the known genes.

CONCLUSIONSThere are obvious differences in gene expression in 801-D-wtp53 after induced by cisplatin than two other controls. It is possible for p53 to regulate the sensitization of lung cancer cells to cisplatin through its downstream target genes.

BACKGROUNDTo construct and express a phage display library of anti human lung cancer monoclonal antibody 5F-11.

METHODSImmunoglobulin variable regions (VH,VL) were amplified from 5F-11 hybridrom by RT-PCR. ScFv genes consisting of VH DNA and VL DNA joined together by a linker DNA were cloned into a phage vector pCANTAB5E. After 4 rounds of screening with lung adenocarcinoma cell line A2 as antigen, an enriched secondary phage display library was obtained.

RESULTSA recombinant phage display library with total of 8×10⁷ pfu/ml was established. Randomized clones from unselected library digested with BstNⅠ showed different patterns, however, those from selected library showed that phages with special pattern were enriched. Twenty-three out of 30 clones were found to respond strongly to A2 cell lines.

CONCLUSIONSThe ScFv of anti-lung adenocarcinoma monoclonal antibody 5F-11 can be successfully produced, which may be useful to widen the application of the antibody.

BACKGROUNDTo study the effects of extraneous p53 antisense RNA on malignant growth and sensitivity to cisplatin of human lung cancer cell line.

METHODS801D cell line with p53 deletion and mutation at 248 codon was selected as a parent cell line. An 1.8 kb human p53 full length cDNA was inserted into a mammalian expression vector PEGFP to construct a p53 antisense RNA recombined plasmid PEGFP-p53(AS) and GFP gene at plasmid was a report gene to monitor extraneous gene expression. The extraneous gene was detected by PCR. The p53 mutation protein was examined by immunohitochemical stain of p53 monoclonal antibody. The inhibition growth efficacy of extraneous p53 in vitro was determined by clonogenic survival assay. Sensitivity of cells to cisplatin was examined with MTT assay. FCM analysis was performed to measure the effect of p53 antisense RNA on cell cycle.

RESULTSTwo cell lines, PEGFP-p53(AS)-801D and PEGFP-801D, were established after transfection of 801-D cells by lipofection and selection. Presence of extraneous p53 gene in PEGFP-p53(AS)-801D was proved by PCR and expression of extraneous p53 was estimated when green fluorescence in those cells was found out under the fluorescent microscopy. Mutated p53 protein in parent cell line 801D was positive and in PEGFP-p53(AS)-801D was negative with immunochemical stain. The inhibition rate of colony formation was 61% for PEGFP-p53(AS)-801D (P < 0.001). The sensitivity of PEGFP-p53(AS)-801D cells to cisplatin was increased. FCM analysis showed that the cell line was arrested at G1 phase.

CONCLUSIONSp53 mutation at 248 code plays an important role on malignant growth and resistance to cisplatin of human lung cancer cell line 801D. Malignant growth of cells with p53 deletion and mutation at 248 codon can be inhibited by extraneous p53 antisense RNA, and simultaneously the sensitivity to cisplatin is also increased.

BACKGROUNDTo investigate the relations between metabolizing enzymes' genetic polymorphism and lung cancer risk in Chinese, especially in heavy smokers.

METHODSCYP1A1, 2D6, 2E1 and GSTM1 genotypes were detected in 180 patients with lung cancer and 224 controls by PCR-based genotype assays.

RESULTSCYP1A1 variant allele, CYP2D6 wild allele, CYP2E1 A genotype, GSTM1-null genotype were found to be associated with lung cancer. The individuals who carried GSTM1-null genotype and one of the CYP1A1, CYP2D6, CYP2E1 'in risk' genotypes had a 2.24-2.69 fold increased risk of lung cancer. The heavy smokers had a significantly increased risk of lung cancer than the non-smokers who carried the same genotype of metabolizing enzymes. The heavy smoker who carried all the four 'in risk' genotypes of metabolizing enzymes had an obviously increased risk of lung cancer (OR=9.85, 95%CI=2.30-45.71).

CONCLUSIONSThe individuals who carry the 'in risk' genotype of metabolizing enzymes have an increased risk of lung cancer. It is positively associated with tobacco carcinogen dose.

BACKGROUNDMatrix metalloproteinase-9 (MMP-9), endostatin (ES) and vascular endothelial growth factor (VEGF) are important angiogenic regulators for many neoplasms. The aim of this study is to judge clinical and prognostic values of detection of serum MMP-9, ES and VEGF in patients with non-small cell lung cancer (NSCLC).

METHODSSerum levels of MMP-9, ES and VEGF were detected in 92 patients with NSCLC, 50 patients with pulmonary benign disease and 52 healthy controls by ELISA method.

RESULTSThe serum levels of MMP-9, ES and VEGF in NSCLC patients were significantly higher than those in patients with pulmonary benign disease and healthy controls (P=0.000, P=0.000, P=0.000). The sensitivity and specificity of serum MMP-9 was 92.51% and 79.10% with a cutoff value of 117.17 μg/L, 88.32% and 74.25% for ES with a cutoff value of 100.31 μg/L, and 83.40% and 75.63% for VEGF with a cutoff value of 380.32 ng/L. Serum MMP-9 and ES levels were significant prognostic factors for lung cancer patients (P=0.0145, P=0.008). The change of serum MMP-9 level after chemotherapy was a useful indicator of prognosis for NSCLC patients (P=0.0322).

CONCLUSIONSThe serum levels of MMP-9, ES and VEGF are significantly increased in patients with NSCLC. They might be used as prognostic parameters in patients with NSCLC.

BACKGROUNDIt was reported that tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was a powerful pulmonary carcinogen, predominantly inducing adenocarcinoma of the lung in mouse. The aim of this study is to assay metabolites of NNK, which are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its O-glucuronide (NNAL-Gluc), and their ratio (NNAL-Gluc/NNAL) in smokers and non-smokers' urine, and to explore the carcinogenicity of NNK among different people.

METHODSUsing high pressure liquid chromatograph (HPLC) and gas chromatograph-mass tadom (GC-MS/MS), NNAL-Gluc and NNAL in 24h urine were detected in 8 healthy smokers, 10 lung cancer smokers and 4 healthy non-smokers.

RESULTSBoth of the two metabolites were not found in non-smokers' urine. The ratios of urine NNAL-Gluc/NNAL were greatly different among different smokers. The mean ratio of NNAL-Gluc/NNAL in healthy smokers was 4.95, and 0.5 in lung cancer smokers.

CONCLUSIONSThe results provide the first evidence for metabolite detection of tobacco-specific nitrosamine in Chinese smokers' urine . The result suggests that detoxification ability of healthy smokers is higher than that of lung cancer smokers. It may provide a detective way to screen high risk people for lung cancer in smokers.