RESUMO
OBJECTIVE: To establish and identify CHO and COS7 cell lines transfected with TIF3cDNA. METHODS: CHO and COS7 cell lines transfected with TIF3cDNA were established with plasmid (pcDNA3.1/V5-His-TOPO) as the vector, using calcium phosphate inductive transfection techniques and G418 cell selection protocols. Expression of the transfected gene was analyzed and identified using western blotting analysis. The non-transfected group and vector-transfected group were designed to compare with the target gene-transfected group. RESULTS: 3 out of 7 CHO cell lines transfected with TIF3cDNA expressed stably high levels of TIF3 encoded protein. Similarly, 2 out of 4 COS7 cell lines had significant over-expression of TIF3 encoded protein. Conclusion Three CHO and two COS7 cell lines transfected with TIF3cDNA have been successfully established. The cell lines are of great value for the future exploration of Cd compounds related oncogenens and for the studies of the other biological functions of the compounds.
Assuntos
Fator de Iniciação 3 em Procariotos/genética , Animais , Células CHO/citologia , Células COS/citologia , Cloreto de Cádmio/toxicidade , Chlorocebus aethiops , Cricetinae , Cricetulus , DNA Complementar/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Fator de Iniciação 3 em Procariotos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
<p><b>OBJECTIVE</b>To study aberrant DNA methylation potentially resulting in changes in the expression of cancer-related genes as a possible epigenetic mechanism for cadmium carcinogenesis.</p><p><b>METHODS</b>Genomic DNA isolated from CdCl(2)-transformed BALB/c-3T3 cells was digested with Mse1 (methylation non-sensitive) alone or with Mse1 and BstU1 (methylation sensitive). The resulting DNA was analyzed for aberrant methylation using PCR-based technique-Methylation-Sensitive Restriction Fingerprinting (MSRF). Several DNA fragments differentially methylated in the transformed cells identified by MSRF were confirmed by Southern hybridization analysis using the aberrantly methylated DNA fragments as the probes.</p><p><b>RESULTS</b>Aberrant DNA methylation was identified in the transformed cells. DNA sequencing and sequence similarity analysis identified one of the aberrantly methylated DNA fragments as the p16 tumor suppressor gene.</p><p><b>CONCLUSION</b>DNA hypermethylation is known to result in gene silencing, it appears that hypermethylation of p16 gene may represent a possible epigenetic mechanism for Cd-induced cell transformation and carcinogenesis.</p>
