Vaults, recently discovered in 1986, are multi-subunit organelles with a molecular mass of ,--,13 MDa. The specific function of vaults is unknown, although they are believed to be involved in internal transport. These ribonucleoproteins are composed of the major vault protein, which comprises ' 70% of the vault's mass, two minor proteins, TEP1 and vPARP, and untranslated RNA(s). It is believed that the protein components of the vault are structural while the RNAs are the functional components. Implications of the vault's involvement in multi-drug resistance in cancer have been made. In some resistant cancer cells, the major vault protein and vRNA(s) are up-regulated up to 15 times when cells are exposed to a cytotoxic drug. Cytotoxic drugs such as doxorubicin are administered as a cancer treatment, but may be ineffective because the drug is actively pumped out of the cell. Multi-drug resistance is the most common failure of chemotherapeutic cancer treatment. In order to prevent the development of multi-drug resistance this research employed the use of small interfering RNA technology to down-regulate the expression of one of the vault RNAs, vRNA3, in cultured GLC4 cells, a small-cell lung cancer cell line. If the vRNA(s) are the functional portion of the vault and a cloned siRNA prevents their up-regulation after drug exposure, the cells should lose their multi-drug resistance, stimulating apoptosis. If successful, this approach may provide an alternative approach to cancer treatment in cells which respond to chemotherapy by increasing the number of vault particles.Initially, the transfection of a plasmid into GLC4 cells was optimized. The best transfection efficiency (N20%) was obtained by using GeneTherapySystems' GenePORTER2 transfection reagent in serum free conditions. To determine if the vault RNAs are the functional portion of the vault complex that confers multi-drug resistance to a cell, a small interfering RNA fragment was designed to specifically knock-down the expression of human vault RNA 3. The siRNA sequence homologous to a portion of vault RNA3 was cloned into an expression vector, and using optimized transfection protocols was transfected into GLC4/REV cells. A Western analysis using caspase-8 antibodies showed no difference in caspase-8 expression in doxorubicin treated and untreated cells. Preliminary results yielded by reverse transcriptase polymerase chain reaction amplification of isolated RNA indicated that the vRNAs were not down-regulated by the siRNAs. / Department of Biology

The gene, cox-1, is over-expressed in cultured GLC4 small cell lung cancer cells concurrent with the development of multi-drug resistance (MDR) as a result of the use of the chemotherapeutic agent used to combat the cancer, doxorubicin. Prevention of MDR has been a tremendous challenge in cancer research and this research is concerned with abolishment of MDR as a cancer survival strategy. RNA-mediated interference technology (RNAi) was employed using siRNA to decrease cox-1 expression and temporarily restore the susceptibility of the cells to doxorubicin. GLC4 cells are of three types: S (sensitive cells never exposed to doxorubicin); ADR (MDR cells cultured in doxorubicin), and; REV (revertant cells previously cultured in presence of doxorubicin but no longer). REV and ADR cells were transfected with cox-1 siRNA. After 24 h, 1x106cells were used for RNA isolation and 1 μg of RNA was used for RT-PCR to assess down-regulation of cox-1 RNA. RT-PCR results indicated that cox-1 RNA was down-regulated to basal levels seen before exposure to doxorubicin. Ct values for GLC4/ADR and cox-1 down-regulated GLC4/ADR cells were 23 and 34, respectively. The result indicated abundant levels and moderate levels of cox-1 mRNA in the ADR cells and the transfected ADR cells respectively. The relative expression level of cox-1 mRNA was 33% higher in the non-transfected GLCR/ADR cells as compared to the transfected GLCR/ADR cells as shown by the curve. Two hundred thousand cells were used for hemacytometer cell counts in the presence of trypan blue to assess cell viability. cox-1 down-regulation in ADR cells resulted in a significantly higher percentage of non-viable cells (25.4%) as compared to its non-transfected control (20.5%) using a Student’s t-test (*P <0.05). Similarly, fluorescence microscopy confirmed that apoptosis was significantly increased in the ADR cells treated with doxorubicin and cox-1 siRNA simultaneously (69.4%) as compared to its non-transfected control (56.7%) (*= P <0.01). A Western blot analysis performed by Fernando Cuadrado indicated that siRNA transfection decreased the expression of COX-1 by 66% in GLC4/ ADR cells as compared to the non-transfected control using densitometry. However, no conclusive results were obtained using flow cytometry as the flow cytometer was incapable of analyzing the mixed cell population (adherent and suspension) which is a characteristic of this cell line, GLC4. Thus, we have clearly demonstrated that MDR cancer cells can be altered temporarily to become susceptible to doxorubicin, a potentially important finding for the treatment of cancer patients. / Department of Biology