| Summary |
In order for a cell to become cancerous, it must go through a number of ordered changes. One of these changes must occur within protein synthesis in order to support cancer cells' increased proliferation. A major factor involved in protein synthesis is eukaryotic translation initiation factor 4G (eIF4G), which binds eIF4E, eIF3, and polyA binding protein (PABP), forming a complex responsible for bringing mRNAs to ribosomes. eIF4G-I has several isoforms that are suggested to be involved specifically in cell proliferation. These isoforms differ at their N-termini, which allows for the binding ofthe factors that recognize mRNA caps and polyA tails with various efficiencies. It is hypothesized that the eIF4G-I isoform levels will differ between non-tumorgenic and tumorgenic cells based on their differences in proliferation. We carried out siRNA knockdown of the longer isoforms to reduce the rate of proliferation in cancer cells and hypothesized that they would become more susceptible to apoptosis when exposed to chemotherapeutic drugs. Western blot analysis confirmed a different representation of these isoforms in tumor-forming vs. non-tumorogenic cell lines. Furthermore, real-time PCR and western analysis was used to verify knockdown of eIF4G-I isoform mRNAs when cells were treated with the corresponding siRNAs. All but one ofthe siRNA targets showed greater than 75% knockdown. Trypan blue proliferation assays and IC50 assays were used to determine the sensitivity to chemotherapy reagents showing that the depletion of eIF4G-1 showed additive effects by a decrease in cell survival when used in conjunction with the reagents etoposide and cisplatin. However, we did not see these additive effects with the analysis of the biscistronic reporter genes that contain an internal ribosomal entry site (1RES), perhaps due to an overall decrease in protein synthesis. The best explanation for these results is a secondary loss from the addition ofthe chemotherapy reagents of supplementary proteins that may be necessary for 1RES induced translation. In conclusion, the additive effects seen with eIF4G-l depletion and the addition ofthe chemotherapy reagents along with the innovative siRNA delivery techniques make eIF4G-l depletion a possible candidate for innovative cancer therapy. |
