Small ubiquitin-like modifiers (SUMO) are a family of proteins that modulate important functional properties,including protein interaction,subcellular localization,protein dimerization,DNA binding and/or transactivation of transcription factors.It has been suggested that SUMO proteins may play an important role in breast carcinogenesis by sumoylation of estrogen signaling proteins such as co-regulators,and breast cancer-related proteins.

We have transferred the different amounts of the plasmid DNA pCGl, which was constructed by cloning rhGM-CSF gene into the eukaryotic expression plasmid pCDS, directly into the BALB/c mice's quadriceps pretreated with bupivacaine. The result of ELISA confirmed that rhGM - CSF was expressed and secreted in mice of group B injected 60?g of plasmid pCGl, but not in mice of group C injected 30?g of plasmid pCGl. The time for the highest level of rhGM-CSF expression was during the fifteenth to twentieth days after plasmid DNA pCGl was injected. Meanwhile, the results of biological activity detection showed that mice's serum of group B (injected 60?g) had the function of retaining the growth of TF - 1 cells. This demonstrates that the rhGM-CSF in mice's serum of group B has certain biological activity. This Model of animal will blaze a new trail in the treatment and prevention of the leukyopenia.

Messenger RNA was isolated directly from lung cancer cell line A549 using magnetic particles. First strand synthesis from mRNA was driven by M-MLV(Moloney Murine Leukemia Virus) reverse transcriptase and random hexam-eric primer, followed by second strand synthesis using RNase H and DNA polymerase I After treatment with T4 DNA polymerase to flush the ends, the double-stranded cDNA was cloned into the plasmid expression vector digested with EcoRI and followed by removing cohesive end. The number of independent clones of the resulting cDNA library was about 9.0 x 105. The estimated percentage of colonies with inserts was about 85 % . The insert size ranges from 0.5 kb ~ 4 kb. The CPP32 gene coding for death protease was obtained by PCR with the cDNA library from lung cancer cells as a template for the first time. Construction of the cDNA library laid a foundation for screening other genes regulating death of lung cancer cells.

GST/MCP-1 fusion protein was overexpressed in Escherichia coli as inculstion bodies. After isolation of the inclusion bodies, the optimum conditions of denaturation and renaturation were studied. The renatured produts were purified to be electroporetically pure by affinity chromatography on immobilised glutathione. The purified product remains biological activities and can react specifically with MCP-1 antibodies by western blot analysis. The in vitro antitumor effect showed that GST/MCP-1 could activate monocytes and lymphocytes to inhibit the growth of a lung adenocarcinoma cell line A549. In vivo, GST/MCP-1 could inhibit the tumor growth in nude mice. These results suggested that MCP-1 had antitumor effect.

Objective To clone E-cadherin and N-cadherin promoters and insert them into a luciferase reporter gene vector, and to characterize the promoter activity of E-cadherin and N-cadherin.Methods E-cadherin and N-cadherin pro-moter were cloned into pGL 4-basic.The resulting plasmids were determined by DNA sequencing .The promoter activity was analyzed in breast cancer cell line ZR 75-1 and hepatocarcinoma cell line HepG 2.Results DNA sequencing showed that the sequences of the cloned promoter regions were correct .Analysis of the reporter gene activity indicated that the E-cad-herin and N-cadherin promoters had the highest transcriptional activity in ZR 75-1 and HepG2 cells.Conclusion The E-cadherin and N-cadherin promoter genes are cloned successfully , contributing much to screening transcription factors that regulate E-cadherin and N-cadherin expression .

In order to investigate if there exists interaction between mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) protein, and how the interaction regulates tumor necrosis factor-? (TNF-?) transcription activity, the human p38 and extracellular-signal regulated protein kinase 2 (ERK2) genes were amplified from human flag-p38 and flag-ERK2 by polymerase chain reaction (PCR) and cloned into pcDNA3-HA. Protein expression of the plasmids was examined by Western blotting. Co-immunoprecipitation was used to identify if there exists interaction between MAPK and STAT3 proteins. If the interaction was approved to be true, report gene system was applied to find how the interaction affect transcriptional expression of TNF-?. After STAT3 pathway was inhibited by RNA interfering, the action on TNF-? activity was determined. The results of DNA sequencing and enzyme digestion showed that the cloned p38 and ERK2 genes were correct, to be 1 080 bp or so. p38 and ERK2 proteins were expressed in 293T cell to be approximately 40 ku. Co-immunoprecipitation data showed that p38 and ERK2 proteins integrated with STAT3 protein in vivo. TNF-? reporter gene activity results found that protein complex of p38-STAT3 and ERK2-STAT3 coordinately increased TNF-? activity. After STAT3 was interfered, the TNF-? activity markedly decreased. These data indicated that there exists interaction between p38 and STAT3 protein, ERK2 and STAT3 protein. The complex of the proteins can coordinately regulate TNF-? expression. After interfereing STAT3 pathway, the coordinated action on TNF-? transcription activity might be obviously reduced.

Objective To construct estrogen receptor α (ERα)trans-activation system. Methods The full length ERα and its different function regions [ ( transcriptional activation function 1 ( AF1 ), DNA inding domain ( DBD), and transcriptional activation function 2 ( AF2 ) ] were amplified from pcDNA3 -ERα by PCR and cloned into the pGAL vector. The expressions of the recombinant plasmids constructed were detected via immunoblotting. The 293T cells transfected with recombinant plasmids of full length ERα, its different function regions and empty vector were divided into 5 groups; each group was divided into 2 parts which were treated with or without estrogen (E2). The transcriptional activity of each group was detected in 293T cells after the recombinant plasmid was co-transfected with 0. 2 μg of estrogen receptor element luciferase(ERE-LUC) and 0. 1 μg of plasmid expressing β-galactosidase and treated with or without 10 nmol/L E2 for 24 hours. Results The full length ERα and its different function regions were expressed in the 293T cells. Compared with the empty pGAL vector, the transcription activities of full length ERα, AF1, AF2 and DBD recombinant plasmids were raised about 20. 44±1.01, 2. 09±0. 11, 8. 09±0. 30 and 1.05±0. 09 fold, respectively, with the induction of E2 after transfection in the 293T colls. Conclusion The trans-activation system of ERα has been successfully established.

Objective To purify and prokaryotically express the histone methyltransferase SET7.Methods The coding sequence of full length SET7 was amplified from breast library by PCR and cloned into the pGEX-KG vector.The correct recombinant plasmid was introduced into E.coli.The expressed protein was identified by SDS-PAGE.Results DNA sequencing indicated the SET7 was constructed.GST-SET7 fusion protein was identified by SDS-PAGE.Conclusion The prokaryotically expressed protein of GST-SET7 is obtained,which will falilitate further study of SET7 function.

Objective To construct the lentiviral vector of RNA interference(RNAi) for DEK,and to detect its effect on breast cancer cell growth.Methods The DEK siRNA was designed and constructed based on DEK sequence using a lentiviral vector.The lentivirul vector containing DEK siRNA was named PSIH-H1-DEK as confirmed by PCR and sequenceing.PSIH-H1-DEK was then packaged with accessory plasmids into lentivirus in 293T cells and selected for 2 weeks with puromycin ( puro ) before the mixed colonies stably expressing DEK siRNA were obtained and the DEK expression was detected by real time PCR( RT-PCR) and Western blotting.The effect of DEK siRNA on ZR75-1 cell growth was determined by cell counting kit.Results Western blot and RT-PCR showed that PSIH-H1-DEK siRNA could suppress DEK gene expression.Suppression of DEK could markedly inhibit the growth of ZR75-1 cells.Conclusion The lentivirus-mediated DEK siRNA is obtained,which will facilitate further research on DEK function in breast cancer development.

AIM: To construct human Egr-1 promoter luciferase reporter system and study its activity induced by i-onizing radiation. METHODS: Egr-1 promoter was obtained by human genomic PCR and cloned into pGL3-basic vector. After transfection of recombinant plasmid into human tumor cells, the Egr-1 promoter activity induced by ionizing radiation was detected by luciferase reporter assay. RESULTS: The luciferasy reporter system of Egr-1 promoter was successfully constructed. The activity of Egr-1 promoter was substantially increased after different doses of IR and reached to the peak at the time point of 48h after IR. CONCLUSION: The Egr-1 promoter was constructed in this study showed IR inducible activity in tumor cells, laying foundation for the research of radiation, mediated gene therapy.