[Establishment and identification of stable transfection of CHO and COS7 cells with TIF3cDNA].

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.

Effect of aberrant DNA methylation on the expression of cancer-related genes in Cd-transformed cells / 中华劳动卫生职业病杂志

<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>

[Antisense TIF3 reverses the oncogenic potential of CdCl2-transformed BALB/c-3T3 cells].

TIF3 (GenBank Accession Number AF 271072) is identified as a novel cadmium- responsive proto-oncogene. In order to determine whether the antisense TIF3 reverses the oncogenic potential of Cd-transformed BALB/c-3T3 cells or not, a stable expression system of CdCl2-transformed BALB/c-3T3 cells with the expression vector containing TIF3 cDNA in the antisense orientation using calcium phosphate and G418 selection protocols is firstly established. Then, the reversal of the oncogenic potential of these cells is tested by soft agar and nude mouse tumorigenicity assay. The results demonstrated that expression of the antisense TIF3 in the CdCl2-transformed BALB/c-3T3 cells results in reversal of the transformed phenotype of the cells. This is evidenced by a 25%-70% decrease in the number of anchorage-independent colonies growing on soft agar and the significant reduced tumorigenic potential of cells in nude mice compared with the corresponding controls. In addition to a significant delay in the onset of appearance of tumors, a significant reduction in size and a 50.8%-55.1% decrease in weight of the tumors are also observed in the mice injected with the TIF3 antisense expressing cells compared with the corresponding controls. The results indicate that antisense TIF3 mRNA expression reverses its oncogenic potential of Cd-transformed BALB/c-3T3 cells and may have therapeutic potential to cancer induced by cadmium.