| BackgroundNon-small cell lung cancer (NSCLC) is one of the most deadly and common malignancy tumors, threatening people's health all over the world. In the past 50 years, the incidence and the mortality of lung cancer rise rapidly, especially in the developed countries. The number of male patients, dying of lung caner, ranks first among all kinds of malignancy tumors. Among the different kinds of NSCLC, the percentage of adenocarcinoma is the largest. However, due to its late-found and fast transfering, there is little chance for most patients to be treated by surgerical methods. What is more sadness is that the chemotherapy and radiotherapy are insensitive to the most patients either. Thus, all the treatment methods of lung cancer are not satisfied in general.Recently, some experts found Angiogenesis in the newborn solid tumors. They thought it's helpful to restrain the growth of tumor by blocking its angiogenesis. This angiogenesis repressed by drugs, will block the blood and supplies to the solid tumor. The drugs, designed according to this theory, have been used into the clinical, such as the Gefitinib and Tarceva (Epidermal Growth Factor Receptor blocker), Cetuximab (the monoclone antibody of EGFR), Bevacizumab (VEGF, vascular endothelial growth factor Blocker). All these drugs have brought great benefits to the patients who are suffering none small cell lung cancer.However, the data-based medicine shows that all these drugs mentioned above were not as sufficient as expected. Late research found that the DLL4/Notch pathway plays a great role during the solid tumor angiogenesis as well. And DLL4/Notch pathway could have interaction with the EGFR,LEF1 and VEGF signal pathway at the crossing-talk point TLE1 during angiogenesis.Therefore, it brings us the following thoughts. Could DLL4 overexpressed in none small cell lung cancer related to the angiogenesis of lung cancer? How could TLE1 mediate the EGFR,LEF1 and DLL4/Notch pathway? What is the structure basis of TLE1 to support the function? Objective1. To detect DLL4 protein expression in 60 cases of none small cell lung cancer (including lung squamous carcinoma and lung adenocarcinoma). To explore the relationship between the expression of DLL4 and clinical charators of none small cell lung cancer.2. To construct theTLE1-Q procaryotic expression system, and express and purify the protein TLE1-Q;3. To obtain the monocrystal of the protein TLE1-Q, explore the structure of the protein TLE1-Q.Methods1. DLL4 protein expression in none small cell lung cancerWe collected 60 lung cancer samples in Changzheng Hospital from Nov. 2007 to Nov.2008. All the samples had been verified as none small cell lung cancer. We tested the DLL4 expression difference among lung squamous carcinoma, lung adenocarcinoma and normal lung tissues by immunohistochemistry methods. We used the SPSS 11.0 to analysis the relationship between the expression of DLL4 and age, gender, histology, differentiation, TNM stage and lymph node metastasis in none small cell lung cancer.2. Construction of theTLE1-Q procaryotic expression system, and expression and purification of the protein TLE1-Q.We reverse-transcripted the mRNA to get the cDNA of the lung adenocarcinoma. The TLE1-Q gene was amplified from this cDNA. The target gene was ligated into the pGEX-4T-1 expression vector which attached a GST tag at the N-terminus of the target protein. Then the recombinant plasmid was transformed into E. coli BL21 (DE3) Codon Plus. The positive clones were cultured in LB. The protein of TLE1-Q were induced at proper time and purified by affinity chromatograph and molecular sieve.3. TLE1-Q monocrystal screening, crystallization optimization, structure model building.We concentrated protein TLE1-Q to 20mg/ml and screened the monocrystal of TLE1-Q by Sitting-drop method. Further-optimizations were carried out according to the pH of the buffer and the concentration of salts of the crystallization buffer. X-ray and Synchrotron Radiation were used to collect the TLE1-Q monocrystal diffraction data, the data were indexed, integrated and scaled to build the TLE1-Q structure model.ResultsPart 1 DLL4 protein up-express in none small cell lung cancer1. The positive rates of the expression of protein DLL4 in none small cell lung cancer was 68.3%.The protein DLL4 was up-expressed in both lung squamous carcinoma, lung adenocarcinoma. The the expression postive rates of DLL4 protein in lung squamous carcinoma and lung adenocarcinoma did not differ statistically (P>0.05).2. The high expression of DLL4 protein in none small cell lung cancer did not correlate with age, gender, histology (P>0.05),but correlated significantly with differentiation, TNM stage and lymph node metastasis (P<0.05).Part 2 Construction of the TLE1-Q procaryotic expression system, expression and purification of the protein TLE1-Q.The sequence of TLE1 N-terminal Q domain obtained by PCR from human lung adenocarcinoma cDNA, was cloned into the prokaryotic expression vector pGEX-4T-1.The sequencing of recombinant plasmid demonstrated that vector pGEX-4T1-TLE1-Q was successfully constructed. Vector pGEX-4T1-TLE1-Q was transformed into E.coli BL21 condon plus.The GST-TLE1-Q (16-136) fusion protein was induced by IPTG, digested by Thrombin, purified with glutathione-sepharose beads and molecular sieve FPLC, identified by SDS-PAGE. The SDS-PAGE showed target protein (14,000 Da) is the interest protein TLE1-Q (16-136), with the purity over 95%.Part 3 Crystallization and optimizationThe crystals used for X-ray diffraction data collection were obtained in 10% v/v tacsimate pH 7.0, 0.1 M HEPES pH 7.4, 2% (w/v) polyethylene glycol 3350, 3% MPD using hanging drops at 4°C in a week, with maximal dimensions of approximately 0.1 x 0.1 x 0.3 mm~3. The complete diffraction data of the native and Se-Met TLE1-Q were collected to 3.5 A and 4.1 A resolution, respectively. The TLE1-Q crystal belonged to orthorhombic crystals in space group C2221, with the unit-cell parameters a = 93.7?, b = 224.1A, c = 161.1A, andα=β=γA=90°. The far-UV circular dichroism spectral deconvolution (CD) analysis and preliminary structure by MAD analysis suggested that secondary structure of TLE1-Q consists of mainlyα-helices (62.5%α-helix, 7.2%β-sheet and 30.3% random coil), showing typical coil-coil secondary structure.ConclusionsIn this research, we found DLL4 shows high expression in none small cell lung cancer (both lung squamous carcinoma and lung adenocarcinoma).This up-expression correlated significantly with differentiation, TNM stage and lymph node metastasis. We cloned, purified TLE1-Q domain (16-136). Furthermore, we crystallized the TLE1-Q domain, the crystal of TLE1-Q domain showed us the preliminary structure of TLE1-Q domain combined with bioinformatics and circular dichroism methods. |
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