Isolation And Identification Of New TPA Responsive Activators Of A New Oncogene NGAL

The neutrophil gelatinase-associated lipocalin(NGAL)gene is a member of lipocalin families. We previously showed that NGAL gene had been overexpressed in the progress of human esophageal epithelial cells transforming to carcinoma, and involved in the malignant invasion and disordered differentiation of the tumor cells. These evidences proved thatNGAL was a new important human oncogene, but its mechanism of transcribed regulation was not clear in the esophageal cancer. Recently we found that the transcription key enhancer element of NGAL gene was loated in the–107 to–82 region in the EC109 cells and this element might be a new TPA responsive element. The activated protein binding with it also might be a new TPA responsive activators that have not been reported previously. Therefore, we will purify and identify the factor to response TPA from esophageal carcinoma cells using some methods incuding DNA affinity chromatography, MOLDI-TOF-MS and analysis of bioinformatics.The key question of this experiment is to purify and identify the new TPA responsive activators of a new oncogene NGAL. If we can identify the TPA responsive activators, we will know what the protein is and how they bind the NGAL promoter region to activate the gene revolution. On this basis, we can manage to confirm that the NGAL is a new target gene of the cancer urged matter TPA; the process that the TPA induce the NGAL outside the cell, the NGAL response in the cell and many biologic molecules participate in it; the excessive express reason of the NGAL in the transformation of oesophagus epithelium cells from normal to malignance.In this experiment, we had compared three chromatographic methods, included the oligonucleotide trapping method, the improved oligonucleotide trapping method and the test tube method. Oligonucleotide trapping method in which a decameric oligonucleotide (AC)5 coupled to Sepharose was used to trap a complex of the transcriptional activators. The complex must be incubated with the NGAL key transcriptional region which has a decameric oligonucleotide (TG)5 for 20 minutes at room temperature. After the transcriptional activators had been adsorbed to the chromatographic medium, eluted with salt to elute the proteins from the column or elute with temperature to elute the proteins bound to the oligonucleotide. In this experiment, we eluted with salt because if we eluted with temperature, the eluted production was DNA-protein complex. The complex should be departed, it is difficult. Firstly, we used 0mol·L-1 NaCl to elute the non specific binding proteins, then eluted with salt solution which the salt concentration was 0.2mol·L-1,0.5mol·L-1 and 1.0mol·L-1 separately. The general eluted volume was 20 column volumes in each group. The eluted solution was Binding buffer which has some salt. The question is that the Tween 20 can not permeate the filter unit and will influence the result of EMSA and silver staining. So in the second method, we used the Hepes·KOH buffer to replace the Binding buffer as a new method. The theory of the test tube method is similar to the improved oligonucleotide trapping method. Added some chromatographic medium coupled with some decameric oligonucleotide (AC)5 to the prepared test tube, then added some complex of the extracted nuclear proteins and NGAL key transcription enhancer element which had a decameric oligonucleotide (AC)5 on both sides. Incubated by circumrotate for 20 minutes at room temperature. After the incubation, eluted the target protein with Hepes·KOH buffer which had salt, the concentration of the salt was 0.2mol·L-1,0.5mol·L-1 and 1.0mol·L-1 separately. After elution, each group was detected by EMSA and silver staining. But for the quantity of target protein was too low, there were not results to the detection,At the purification of the improved oligonucleotide trapping method, we discovered that the group 0.2mol·L-1 and 0.4mol·L-1 had the target proteins after every eluted group were detected by EMSA. On this basis, we silver stained the two group samples, and selected and cut 4 suspicious strips in the 0.2mol·L-1 group and 5 suspicious strips in the 0.4mol·L-1 group to be identified by MALDI-TOF-MS. Analyzed the MS datum with bioinformatics, we found that there were more than 20 proteins for each silver stained strip. Then analyzed with the strip molecular weight, we preliminary selected 12 possible proteins for the further research, and most of these proteins were not reported as TPA reaction.In conclusion, the improved oligonucleotide trapping method is the best method for purify the NGAL new TPA responsive activators; EMSA is a mostly important means to supervise in the purified process; Analyzed with bioinformatics and MALDI-TOF-MS, we preliminary selected 12 possible proteins for the further research, and most of these proteins were not reported as TPA responsive activators.