RESUMO
The recombined adenovirus DNA was transfected into 293 cells for packing and amplification of replication-deficient Ad-CMV-E6/E7, Ad-K14 -E6/E7 virus was purified by CsCl density gradient centrifugation , recombined adenovirus Ad-CMV-E6/E7, Ad-K14 -E6/E7 were used as experimental group, while pAd-CMV and pAdtrack-K14 were used as control group. Four of them were injected through one main vein of nude mice tail respectively. These mice were then treated with 0.05 mg 17beta-estradiol over 12 weeks. Mice were anaesthesiaed with 2.5% Avertint and the vagina, mammary gland, ovaries and uterus were dissected and fixed in 3.75% paraformaldehyde overnight at 4 degrees C. Paraffin-embedded sections, HE staining and identification of P53 and Bcl-2 protein via immunohistochemistry were performed. The expression of E6/E7 was verified by RT-PCR in different tissue of nude mice. HE staining showed evident hyperplasy in cervix-uterus transformation zone of experimental group 2. The expression of mutant P53 and Bcl-2 were higher than control group via immunohistochemical S-P method in uterus stroma-cell. Western blotting also showed that E6 protein was expressed. The expression of E6/E7 was higher than control group by human cytokeratin promoter 14 and hyperlasy changes were detected in epithelial tissue of cervix-uterus transformation zone.
Assuntos
Animais , Feminino , Humanos , Camundongos , Adenoviridae , Genética , Western Blotting , Linhagem Celular , Doenças dos Genitais Femininos , Patologia , Virologia , Genitália Feminina , Patologia , Virologia , Imuno-Histoquímica , Glândulas Mamárias Animais , Metabolismo , Patologia , Camundongos Nus , Proteínas Oncogênicas Virais , Genética , Metabolismo , Ovário , Metabolismo , Patologia , Papillomaviridae , Metabolismo , Fisiologia , Proteínas E7 de Papillomavirus , Proteínas Proto-Oncogênicas c-bcl-2 , Metabolismo , Proteínas Repressoras , Genética , Metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53 , Metabolismo , Útero , Metabolismo , Patologia , Vagina , Metabolismo , PatologiaRESUMO
In this study, we used an adenoviral vector -melanoma differentiation-associated gene-7 [Ad-mda7] to examine the effect of the ectopic production of MDA-7/IL-24 on cell migration and invasion by human cervical cancer cells. The study took place in the Department of Biochemistry and Molecular Biology, Chongqing Medical University, Chongqing, China, between April 2006 and November 2006. The change of metastasis of cervical cancer cells [CaSki] cells were detected by Cell Migration Assay and Cell Invasion Assay after treated with Ad-mda7. The production of proteins associated with cell migration and invasion were detected by western blot. Cervical cancer cells treated in vitro with Ad-mda7 migrated and invaded less than cells treated with phosphate-buffered saline [PBS] or Ad-Luc [vector control]. Melanoma differentiation-associated gene-7 /IL-24 inhibited migration and invasion by down-regulating the production of matrix metalloproteinase-2 [MMP-2] and by up-regulating the production of p38 mitogen-activated protein kinase. relative to PBS and Ad-Luc. These results show that MDA-7/IL-24 inhibits invasion and migration by cervical cancer cells by down- or up-regulating proteins associated with these processes, resulting in reduced metastasis. Thus, Ad-mda7 should be considered a therapeutic agent that can inhibit primary tumor growth and prevent metastasis
RESUMO
<p><b>OBJECTIVE</b>To analyze the transcription activation and possible regulation mechanism of human X-box binding protein 1(XBP1)gene 5'upstream DNA sequence in different cell lines.</p><p><b>METHODS</b>Six kinds of XBP1 promoter deletion mutants were cloned into pGEM-Teasy vector, which included XBP1 gene 5' upstream -1039 to 66 bp,-859 to 66 bp,-623 to 66 bp,-351 to 66 bp,-227 to 66 bp,-227 to -45 bp respectively. Every deletion mutant sequence was cut from Teasy-XBP1p by KpnI and Xho I, and subcloned into pCAT3-Basic to produce a set of constructs termed as p1-XBP1p, p2-XBP1p, p3-XBP1p, p4-XBP1p, p5-XBP1p, p6-XBP1p, respectively. The transcription activity of each construct was detected after transiently transfecting K562, HepG2,NIH-3T3 and L0(2)cell with FuGENE 6 transfection reagent. Cells transfected by pCAT3-Basic or pCAT3-Promoter were used as negative and positive controls. The activity of chloramphenicol acetyltransferase(CAT), which reflects the transcription activation of the XBP1 gene promoter, was detected by ELISA after 48 hours of transfection.</p><p><b>RESULTS</b>The reporter vectors of six kinds of XBP1 promoter deletion mutants were successfully constructed, as confirmed by restriction enzyme digestion and sequencing. The activities of p4-XBP1p and p5-XBP1p were higher than the other deletion mutants in K562 and HepG2. And the activity of p5-XBP1p was the highest in HepG2. There was no activity detected from any transfected NIH-3T3.</p><p><b>CONCLUSION</b>The XBP1 gene promoter can transactivate its downstream gene to transcription. The core sequence of XBP1 promoter was implied between -227 bp and 66 bp. This sequence was connected with the transcriptional activity of XBP1 promoter closely. Its transcription activity varies with different cell lines. XBP1 promoter might drive gene expression with cell-type specificity.</p>