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Wang H.,Beijing TB and Thoracic Tumor Research Institute | Lai B.,Beijing TB and Thoracic Tumor Research Institute | Li W.,Beijing TB and Thoracic Tumor Research Institute | Yang X.,Beijing TB and Thoracic Tumor Research Institute | And 3 more authors.
Chinese Journal of Lung Cancer | Year: 2010

Background and objective: 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. Methods: 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. Results: 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. Conclusion: 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.


PubMed | Beijing TB and Thoracic Tumor Research Institute
Type: Journal Article | Journal: Zhongguo fei ai za zhi = Chinese journal of lung cancer | Year: 2010

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.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.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.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.


PubMed | Beijing TB and Thoracic Tumor Research Institute
Type: Journal Article | Journal: Zhongguo fei ai za zhi = Chinese journal of lung cancer | Year: 2010

Lung cancer is one of the most common cancers worldwide and causes more deaths per year than other cancers. Autoantibody was one hotspot in tumor marker research. But the clinical value of Survivin autoantibody is not clear in lung cancer patients at present. The aim of this study is to determine whether Survivin autoantibody could be used as a diagnostic factor of lung cancer and what the clinical value of Survivin autoantibody was in nonsmall cell lung cancer (NSCLC).Survivin cDNA sequence was obtained by RT-PCR and inserted into prokaryotic expression vector pET30a(+). Fusion protein of Survivin was expressed in E. coil BL21(DE3). SDS-PAGE and Western blot were used to confirm the fusion protein. The Survivin protein was purified by Ni-NTA agarose affinity chromatography. At last, indirect ELISA was used to detect Survivin autoantibody level in the serums of 215 samples from NSCLC patients and serums from 20 samples of nonmalignant lung disease patients as well as 89 samples of healthy persons were also detected as control.The recombinant vector pET30a(+)/Survivin was contructed and the Survivin protein is successfully expressed in E.coil BL21(DE3) as inclusion body. Indirect ELISA was done to detect Survivin autoantibody in these serums using purified Survivin protein. It was shown that the positive rate of Survivin autoantibody was 19.5%, with a specificity of 88.9% in NSCLC patients. The expression of Survivin autoantibody has correlation with the volume of tumor and the metastasis of tumor in NSCLC patients (P < 0.05). In our research, positive detection rate of NSCLC was much improved by detecting Survivin autoantibody combined with CEA compared to other tumor markers combined with CEA.In this study, purified fusion protein of Survivin was successfully obtained. Indirect ELISA for detecting Survivin autoantibody in serum of NSCLC patients using purified Survivin protein was established. Our results indicated that Survivin autoantibody as a latent value of tumor marker could be used for clinical diagnosis in NSCLC patients.


PubMed | Beijing TB and Thoracic Tumor Research Institute
Type: Journal Article | Journal: Zhongguo fei ai za zhi = Chinese journal of lung cancer | Year: 2010

It has been proved that mTOR was an important signal transduction molecular and protein kinase regulating cell growth and proliferation, and mTOR could activate the downstream protein effector. PTEN could negatively regulate mTOR signal pathway and inhibit its activity. The aim of this study is to detect the mRNA expression levels of mTOR and PTEN gene, which are the key genes of mTOR signaling pathway in human non-small cell lung cancer (NSCLC) tissue. The relationship between mTOR signaling pathway and NSCLC is also explored.Lung cancer tissue specimens were obtained from 65 patients. Adjacent-tumor non-small cell lung cancer tissues from the 30 patients were served as control. The RT-PCR technique was used to detect the mTOR and PTEN gene expression levels.The average mRNA expression levels of mTOR gene were significantly higher (0.23 +/- 0.16) in lung cancer than in adjacent-tumor tissue (0.12 +/- 0.09)(P < 0.01). The average mRNA expression levels of PTEN gene were (0.19 +/- 0.28) in lung cancer, while the mRNA expression levels of PTEN gene were (0.53 +/- 0.28) in adjacent-tumor tissue (P < 0.01). The levels of PTEN gene expression in non-small cell lung cancer were significantly lower than that in adjacent-tumor lung tissue. There are not significant relationship between mTOR and PTEN gene expression levels and patients age, gender, pathological type, differentiation, lymph node metastasis, except tumor size.The expression of mTOR is activated in NSCLC. The expression of PTEN is absent or decreased. The mTOR activated in NSCLC may be correlate with the absent or decreased of PTEN. The absent or decreased expression of PTEN and the actived mTOR may play important roles in carcinogenesis and metastasis of NSCLC.


PubMed | Beijing TB and Thoracic Tumor Research Institute
Type: Journal Article | Journal: Zhongguo fei ai za zhi = Chinese journal of lung cancer | Year: 2010

Autoantibodies as new tumor markers may play an important role in the early diagnosis and evaluating the prognosis of lung cancer. In this study, we detect epidermal growth factor receptor (EGFR) autoantibodies using peptide array and screen the epitopes which are recognized by EGFR autoantibodies.Peptide array covering the extracellular domain of EGFR protein was synthesized by a synthesizer (ASPSL) made by Intavis company. EGFR autoantibodies in the serums of non-small cell lung cancer patients was detected using peptide array.Six of 20 patients were found to have EGFR autoantibodies. The positive rate is 30%. Nine high frequency spots were found in the 6 positive patients and 8 high frequency spots clustered in the III and IV domains.These findings will offer new clues for the further studies of EGFR and EGFR autoantibodies.


PubMed | Beijing TB and Thoracic Tumor Research Institute
Type: Journal Article | Journal: Zhongguo fei ai za zhi = Chinese journal of lung cancer | Year: 2010

Currently, only a limited numbers of tumor markers for non small lung cancer (NSCLC) diagnosis, new biomarker, such as serum autoantibody may improve the early detection of lung cancer. Our objective is construction human lung squamous carcinoma and adenocarcinoma T7 phage display cDNA library from the tissues of NSCLC patients.mRNA was isolated from a pool of total RNA extract from NSCLC tissues obtained from 5 adenocarcinomas and 5 squamous carcinomas, and then mRNA was reverse transcribed into double stranded cDNA. After digestion, the cDNA was inserted into T7Select 10-3 vector. The phage display cDNA library was constructed by package reaction in vitro and plate proliferation. Plaque assay and PCR were used to evaluate the library.Two T7 phage display cDNA library were established. Plaque assay show the titer of lung squamas carcinoma library was 1.8*10(6) pfu, and the adenocarcinoma library was 5*10(6) pfu. The phage titer of the amplified library were 3.2*10(10) pfu/mL and 2.5*10(10) pfu/mL. PCR amplification of random plaque show insert ratio were 100% (24/24) in adenocarcinoma library and 95.8% in human lung squamas carcinoma library (23/24). Insert range from 300 bp to 1 500 bp.Two phage display cDNA library from NSCLC were constructed.


Ma L.,Beijing TB and Thoracic Tumor Research Institute | Yue W.,Beijing TB and Thoracic Tumor Research Institute | Zhang L.,Beijing TB and Thoracic Tumor Research Institute | Wang Y.,Beijing TB and Thoracic Tumor Research Institute | And 2 more authors.
Chinese Journal of Lung Cancer | Year: 2010

Background and objective: Lung cancer is one of the most common cancers worldwide and causes more deaths per year than other cancers. Autoantibody was one hotspot in tumor marker research. But the clinical value of Survivin autoantibody is not clear in lung cancer patients at present. The aim of this study is to dertermine whether Survivin autoantibody could be used as a diagnostic factor of lung cancer and what the clinical value of Survivin autoantibody was in non-small cell lung cancer (NSCLC). Methods: Survivin cDNA sequence was obtained by RT-PCR and inserted into prokaryotic expression vector pET30a(+). Fusion protein of Survivin was expressed in E.coil BL21(DE3). SDS-PAGE and Western blot were used to confirm the fusion protein. The Survivin protein was purified by Ni-NTA agarose affinity chromatography. At last, indirect ELISA was used to detect Survivin autoantibody level in the serums of 215 samples from NSCLC patients and serums from 20 samples of nonmalignant lung disease patients as well as 89 samples of healthy persons were also detected as control. Results: The recombinant vector pET30a(+)/Survivin was contracted and the Survivin protein is successfully expressed in E.coil BL21(DE3) as inclusion body. Indirect EILSA was done to detect Survivin autoantibody in these serums using purified Survivin protein. It was shown that the positive rate of Survivin autoantibody was 19.5%, with a specificity of 88.9% in NSCLC patients. The expression of Survivin autoantibody has correlation with the volume of rumor and the metastasis of tumor in NSCLC patients (P<0.05). In our research, positive detection rate of NSCLC was much improved by detecting Survivin autoantibody combined with CEA compared to other tumor markers combined with CEA. Conclusion: In this study, purified fusion protein of Survivin was successfully obtained. Indirect ELISA for detecting Survivin autoantibody in serum of NSCLC patients using purified Survivin protein was established. Our results indicated that Survivin autoantibody as a latent value of tumor marker could be used for clinical diagnosis in NSCLC patients.


PubMed | Beijing TB and Thoracic Tumor Research Institute
Type: Journal Article | Journal: Zhongguo fei ai za zhi = Chinese journal of lung cancer | Year: 2010

Currently, there are only limited number of diagnostic tumor markers for non-small-cell lung cancer (NSCLC). The aim of this study is to screen and identify non-small-cell lung cancer associated antigens, by using of cancer sera containing antibodies which react with autologous cellular antigens (tumor-associated antigens TAAs), which can be used in NSCLC early screening or target treatment.Two T7 phage display cDNA libraries were panning against the sera from 24 patients with NSCLC. Then the enriched libraries were screened with the pool of sera from NSCLC patients, the positive clones encoding antigenic genes were obtained after screening, and the nucleotide sequences of positive were identified and analyzed with BLAST software in GenBank.After immunoscreen two T7 phage display cDNA libraries with serum from patients with NSCLC, thirty-one positive clones were obtained and sequence results showed they were derive from 15 genes, 12 of 15 genes were homologous to the genes known in GenBank, such as RHOA, ITGB4, and HNRNPA2/B1 et al . The other three genes expressed sequence tags (ESTs) were not found in GenBank.Fifteen NSCLC associated antigen genes and three candiated gene were obtained by SEREX from T7 phage display cDNA libraries.


PubMed | Beijing TB and Thoracic Tumor Research Institute
Type: Journal Article | Journal: Zhongguo fei ai za zhi = Chinese journal of lung cancer | Year: 2010

It has been known that abnormality of PI3K/Akt/mTOR signal pathway plaied an important role in the oncogenesis of lung cancer. In this study, we tried to detect the mRNA expression levels of VEGF, PI3K, Akt, mTOR gene, the key genes of PI3K/Akt/mTOR signal pathway, in human non-small cell lung cancer (NSCLC) tissue and explore the relationship between PI3K/Akt/mTOR signaling pathway and non-small cell lung cancer.Lung cancer tissue specimens were obtained from 40 patients. Adjacent-tumor NSCLC tissues from the 30 patients were served as control. The RT-PCR technique was used to detect the VEGF, PI3K, Akt, mTOR gene expression levels.The average mRNA expression levels of VEGF, PI3K, Akt, mTOR gene in lung cancer were (40+/-59)%, (61+/-23)%, (77+/-32)% and (43+/-21)% respectively, while the mRNA expression levels of VEGF, PI3K, Akt, mTOR genes in adjacent-tumor tissue were (16+/-40)%, (23+/-16)%, (10+/-12)% and (20+/-17)%, respectively. All the levels of VEGF, PI3K, Akt, mTOR gene expression in NSCLC were significantly higher than that in adjacent-tumor lung tissue (P <0.01).Our preliminary data have demonstrated that PI3K/Akt/mTOR signaling pathway is activated in the tumor cells of NSCLC. The activated PI3K/Akt/mTOR signaling pathway might be an important role in the pathogenesis of NSCLC.


PubMed | Beijing TB and Thoracic Tumor Research Institute
Type: Journal Article | Journal: Zhongguo fei ai za zhi = Chinese journal of lung cancer | Year: 2010

Serum autoantibody detection is useful means for the early diagnosis and prognosis of cancer. So our objective was to synthesize peptide array to analyse p53 autoantibodies in the sera of patients with non small cell lung cancer (NSCLC).Cellulose-bound overlapping peptides (12 mers) derived from p53 wild type protein were synthesized using SOPTs synthesis technique by an AutoSpot robot -ASP SL (Intavis, Germany). The membrane was incubated with 1/400 dilutions of p53 monoclonal antibody (Sc-53394) to establish a new approach to detect p53 antibody, and the epitopes of the p53 monoclonal antibody is already known. We analysed the p53 autoantibodies from the sera of NSCLC and controls by peptide array and ELISA.We synthesized on cellulose membranes twelve-amino-acid overlapping peptides which included all of the sequences of the polypeptide chain of p53. The p53 autoantibody was positive in seven cases of thirty patients sera with NSCLC and was negative in sera of the controls, with the same result of ELISA CONCLUSIONS: The peptide array could be applied not only to detect the autoantibodies in the sera of patients with lung cancer, but also to map the epitopes of the autoantibodies which might be useful for the early diagnosis and prognosis of cancer.

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