5-FU is a known nuclear export inhibitor and has been used as a chemotherapy. 5-FU inhibits nuclear export by increasing the permeability of the nuclear pores which allows the protein Ran to leak out of the nucleus. Ran is a protein which is necessary for nuclear export. Ran(GTP) helps exportin bind to the NES on cargo proteins, without ran to facilitate cargo and exportin binding proteins are not able to be exported from the nucleus.
Bortezomib is a known proteasome inhibitor of 26s. Proteasomes are large protein complexes which are responsible for the degradation of ubiquitinated proteins. The proteasomes break peptide bonds degrading proteins back down to simple amino acids so they can then be recycled and used in the creation of new proteins.
The protein p53 is a transcription factor which when it accumulates in high levels in the nucleus causes cell apoptosis. The protein p53 is taken out of the cell by the protein mdm2. Four mdm2 proteins bind to p53 which causes the NES to be shown allowing for nuclear export and then degradation.
By inhibiting the nuclear export of p53 and then inhibiting the degradation of p53 through 5-FU and Bortezomib treatment it would be possible to induce apoptosis in these cells. Additionally preventing nuclear export and protein breakdown in these cells can break multiple pathways inhibiting homeostasis leading to the induction of apoptosis.
He La Cells were treated for 6 hours in 4 different groups. Untreated for the control, 5-F, Bortezomib or 5-FUand Bortezomib.
Coverslips were washed with 1xPBS, then fixed with a 3% formaldehyde. The coverslips were washed with 1xPBS wash and permeabilized with 0.5% Triton X-100. The Coverslips were washed with 1xPBS wash and blocked with 5% FBS. Ran specific or p53 specific Primary antibodies were fixed to the sample using the Hybridization Chamber. The coverslips were washed with 1xPBS wash then secondary antibodies were fixed to the sample using the Hybridization Chamber. The coverslips were washed with 1xPBS wash then stained with DAPI. Samples were then permanently secured to the coverslip by using nail polish to secure the coverslip onto a labeled glass slide. Then Immunofluorescence imaging was done by using the BioRAD ZOE Microscope.
Pictures were analyzed using Image J software. Using the freehand tool, for each picture 25 nuclei were outlined and data points for area, Int Density, and Raw-In-Den was recorded. Also, for each picture, 3 rectangles of the background were captured, the background mean was recorded and averaged to find the background mean of that picture. Next nuclear fluorescence was calculated for each nucleus by subtracting the product of the area of the nucleus and the background mean of that picture from the Int Density of each nucleus. For each picture nuclear fluorescence values were averaged to find the average nuclear fluorescence. Next the standard error was calculated by dividing the average by the square root of samples averaged (25 in this case). Lastly the average of nuclear fluorescence of the 5-FU was divided by the control then imputed into bar graphs to better visualize the results of the experiment with a labeled section to compare all groups with p53 or Ran stain.
Treatment of HeLa cells with 5-FU, Bortezomib, and both resulted in a decrease in nuclear ran. Treatment of HeLa cells with 5-FU and Bortezomib resulted in a decrease in nuclear p53, and treatment with both resulted in a doubling of nuclear p53.
Ran Immunofluorescence of He La Cells treated for 6 hours with Untreated, 5-FU, Bortezomib, and 5-FU and Bortezomib. The percentage of average nuclear Ran fluorescence compared to the control determined using images. Sample sizes were untreated (50), 5-FU (50), Bortezomib (100), and 5-FU and Bortezomib (100). The error bars represent standard error.
p53 Immunofluorescence of He La Cells treated for 6 hours with Untreated, 5-FU, Bortezomib, and 5-FU and Bortezomib. The percentage of average nuclear p53 fluorescence compared to the control determined using images. Sample sizes were untreated (50), 5-FU (50), Bortezomib (75), and 5-FU and Bortezomib (94). The error bars represent standard error.
Bortezomib had a similar impact to 5-FU on nuclear ran levels. In our experiment there was an 80 % reduction in nuclear RAN levels in cells treated with Bortezomib for 6 hours compared to the untreated cells. This supports the hypothesis that Bortezomib decreases nuclear RAN levels. Bortezomib’s known interaction in cells is to prevent the proteasome from breaking down proteins. Bortezomib is known to decrease efficiency of the proteasome. Nuclear RAN levels may have lowered because the cell may have sensed that proteins are no longer being broken down and stopped producing many proteins in order to prevent apoptosis from triggering due to a build up of proteins, or cell resources were used to produce more proteasomes and RAN expression was lowered. Another option may be that RAN may play another role in the cell and has been relocated in the cytoplasm to aid in breaking down proteins or making new proteasomes. Another option is that bortezomib may play another function in the cell other then stopping proteasomes from working.