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<p><b>BACKGROUND AND OBJECTIVE</b>The p53 as a transcription factor in cell stress was activated to regulate cell cycle and programmed cell death to inhibit tumor growth. Usually, p53 is kept in non-activated state through various mechanisms, including the action of p53 C-terminal negative regulatory sequences. The purpose of the study is to prepare the two types p53 recombinant adenoviruses that carry full-length p53 as well as deletion of negative regulatory sequences at p53 C-terminus and to detect exogenous GFP expression in human lung cancer cell infected-virus by FCM scatter plot.</p><p><b>METHODS</b>Using pAdEasy-Track vector system the p53 recombinant plasmids was constructed and the homologous recombinants in E. coli was produced. The three kinds of recombinant adenovirus in L293 cells was generated, sequencing proved. Exogenous GFP expression in human lung cancer 801D cells infected-virus was detected by FCM scatter plot.</p><p><b>RESULTS</b>p53 recombinant adenoviruses named Ad-p53(wtp), Ad-p53(del) and Ad-(empty carrier) were produced. Results of sequences indicate that the Ad-p53(del) was deletion of 111 bases before stop codon TGA and of 3 untranslated region at p53, the Ad-p53(wtp) no loss of any p53 base, the Ad-(empty carrier) no p53 sequence. FCM scatter plot indicate the percentage of 801D cells expressed GFP with three kinds of viral infection was almost same and was increased with the virus density. 801D contains ratio of cells with different fluorescence intensity.</p><p><b>CONCLUSION</b>The preparation of recombinant adenovirus, Ad-p53(del), pA-p53(wtp) and Ad-(empty carrier). The cells expressed-GFP can be quantitatively detected by FCM scatter plot. It was provide that the reliability of the virus system and accurate method for selecting viruses density to infecting cells.</p>
Subject(s)
Animals , Humans , Mice , Adenoviridae , Genetics , Flow Cytometry , Methods , Genes, p53 , Green Fluorescent Proteins , Genetics , Recombination, GeneticABSTRACT
<p><b>BACKGROUND</b>Matrix 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).</p><p><b>METHODS</b>Serum 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSIONS</b>The 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.</p>
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<p><b>BACKGROUND</b>Tobacco-specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most important carcinogen in cigarette. Models induced by NNK are widely used in investigations about the mechanisms of pulmonary neoplasia and chemoprevention studies. The aim of this study is to explore the pulmonary precancerous lesions induced by NNK and its possible mechanisms.</p><p><b>METHODS</b>Fifteen Wistar rats were divided into two trial groups, in which the high-dose group was instilled with iodized oil including 10 mg (50 mg/kg) NNK into the left lower lobar bronchus, and the low-dose group received 5mg ( 25mg/kg) NNK. Another 15 Wistar rats were instilled only with iodized oil as control group. All rats were examined immediately after instillation and followed up periodically by pulmogram. The pulmonary tissues of rats were pathologically examined, and the expression of AE1/AE3, PCNA and p53 was detected by immunohistochemical method.</p><p><b>RESULTS</b>The pulmograms showed that the iodized oil localized at the bottom of left lobe and disappeared 107 days later. In trial group, 10 of 15 rats (67%) had nodus at the bottom of left lobe. All of rats in trial group (15/15) displayed atypical hyperplasia in alveolar region, showing single or multiple layers of proliferative epithelial cells along intact alveolar septa with irregular and non-discrete margins of lesion, but continuous alveolar spaces were not obliterated by proliferative epithelial cells. Ten of 15 rats in trial group showed severe atypical hyperplasia of glandular epithelium with occasional infiltrating to muscular layer. All of those atypical hyperplasia cells showed positive AE1/AE3 expression. The positive rate of PCNA was 90% (9/10) and 100% (5/5) in low-dose group and high-dose group respectively, which was significantly higher than that in control group (13%, 2/15) (P=0.000, P=0.001). The positive rate of p53 expression was 50% (5/10) and 60% (3/5) in low-dose group and high-dose group respectively, which was significantly higher than that in control group (0) (P=0.005, P=0.009). However, there was no remarkable difference in PCNA and p53 expression between low-dose group and high-dose group (P > 0.05).</p><p><b>CONCLUSIONS</b>Transbronchial instillation of iodized oil including tobacco-specific NNK can induce pulmonary lesions as atypical hyperplasia of alveolar cell and glandular epithelium in Wistar rats. This model can be used in experimental studies about tobacco-related lung cancer.</p>
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<p><b>BACKGROUND</b>The 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.</p><p><b>METHODS</b>801D 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.</p><p><b>RESULTS</b>Clone 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.</p><p><b>CONCLUSIONS</b>BVP22 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.</p>
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<p><b>BACKGROUND</b>It 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.</p><p><b>METHODS</b>Using 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.</p><p><b>RESULTS</b>Both 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.</p><p><b>CONCLUSIONS</b>The 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.</p>
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<p><b>BACKGROUND</b>Cytochrome P450 2A6 (CYP2A6) plays an important role in oxidation of nicotine and in activation of tobacco-related carcinogens. It has been suggested that individuals with defective CYP2A6 allele are at a lower risk of developing lung cancer. This study is to investigate the frequency of CYP2A6 gene deletion and the relationship of CYP2A6 genetic polymorphism with lung cancer risk in Chinese.</p><p><b>METHODS</b>A case-control study which detected CYP2A6 genotype of 180 patients with lung cancer and 224 controls by PCR-based genotype assay was conducted.</p><p><b>RESULTS</b>No relationship was found between the frequency of CYP2A6 gene deletion and lung cancer risk. There was only one case of CYP2A6 del/del genotype in the controls. The frequency of CYP2A6 del allele was 13.8% in the controls, and 12.8% in lung cancer cases. The CYP2A6 del/del genotype was not found in lung cancer cases.</p><p><b>CONCLUSIONS</b>There is no difference in frequency of CYP2A6 gene deletion between lung cancer cases and controls.</p>
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<p><b>BACKGROUND</b>With 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>5F11-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.</p><p><b>CONCLUSIONS</b>The killing efficacy of 5F11-DOX on human lung adenocarcinoma cell line A2 is obviously higher than that of the free DOX.</p>
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<p><b>BACKGROUND</b>To investigate the relations between metabolizing enzymes' genetic polymorphism and lung cancer risk in Chinese, especially in heavy smokers.</p><p><b>METHODS</b>CYP1A1, 2D6, 2E1 and GSTM1 genotypes were detected in 180 patients with lung cancer and 224 controls by PCR-based genotype assays.</p><p><b>RESULTS</b>CYP1A1 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).</p><p><b>CONCLUSIONS</b>The 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.</p>
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<p><b>BACKGROUND</b>To 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).</p><p><b>METHODS</b>Total 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.</p><p><b>RESULTS</b>Six 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.</p><p><b>CONCLUSIONS</b>There 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.</p>
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<p><b>BACKGROUND</b>To study the effects of extraneous p53 antisense RNA on malignant growth and sensitivity to cisplatin of human lung cancer cell line.</p><p><b>METHODS</b>801D 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.</p><p><b>RESULTS</b>Two 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.</p><p><b>CONCLUSIONS</b>p53 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.</p>
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<p><b>BACKGROUND</b>To construct and express a phage display library of anti human lung cancer monoclonal antibody 5F-11.</p><p><b>METHODS</b>Immunoglobulin 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.</p><p><b>RESULTS</b>A 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.</p><p><b>CONCLUSIONS</b>The ScFv of anti-lung adenocarcinoma monoclonal antibody 5F-11 can be successfully produced, which may be useful to widen the application of the antibody.</p>
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<p><b>BACKGROUND</b>To study the inhibition effects of both extraneous right sense and antisense p53 on malignant phenotype of human lung cancer cell line.</p><p><b>METHODS</b>The named 801D cell line with p53 deletion and mutation at 248 code was selected as a model in vitro. The recombined plasmid pEGFP-p53(RS) and pEGFP-p53(AS) were constructed. The extraneous gene was detected by PCR. The p53 mutation protein was examined by immunohistochemical stain of p53 antibody. The inhibition effect of extraneous p53 on tumor growth in vitro were determined by clonogenic survival assay. FCM analysis was carried out in cells. The inhibition effect on malignant growth of extraneous p53 in vivo was observed by heteroplastic transplant on nude mouse.</p><p><b>RESULTS</b>The transfected cell lines, pEGFP-p53(AS)-801D, pEGFP-p53(RS)-801D and pEGFP-801D were established. Presence of extraneous p53 and neo genes in pEGFP-p53(AS)-801D and pEGFP-p53(RS)-801D was proved by PCR and green fluorescence was found out in those cells under the microscope. Mutant protein in pEGFP-p53(AS)-801D was negative by immunohistochemical stain. The malignant growth of these transfected cell lines was inhibited comparing with parents in vivo and in vitro. Inhibition rate of colony formation was 62.0% for pEGFP-p53(AS)-801D and 80.8% for pEGFP-p53(RS)-801D. The tumorigenicity in nude mice was suppressed. Inhibition effects of extraneous right sense p53 on malignant growth of 801D was more distinct. FCM analysis showed that pEGFP-p53(AS)-801D cells were arrested at G1 phase.</p><p><b>CONCLUSIONS</b>The transfected cell lines with extraneous right sense and antisense p53 appear that malignant growth can be inhibited in vivo and in vitro.</p>
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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.\;[