Progress 01/01/06 to 12/31/07
OUTPUTS: Inappropriate expression of Cox-2 is believed to contribute to both tumorigenesis as well as resistance of tumor cells to standard therapeutics. A commercially available Cox-2 inhibitor (deracoxib, Novartis) will be used to determine if Cox-2 inhibition increases the sensitivity of melanoma cells to radiation induced death. We found that our cell lines do express Cox-2 mRNA and protein by using RT-PCR and Western Blotting. We have completed the clonogenic assay work for each of the cell lines with 24 hours drug exposure. We did not find any differences in the cell survival curves between the drug and non-drug exposed cells when irradiated. To see if this was due to continued expression of Cox-2 or inadequate blockade of the pathway we tested prostaglandin levels in the cell culture media and found that there was still prostaglandin at 24 hours but with continued exposure of the cells to drug we could get prostaglandin levels to decrease. We have repeated the clonogenic assay in the cell lines with a longer drug exposure and found no increase in killing indicating at least in vitro that Cox-2 inhibition does not have a large effect on melanoma cell survival. We have looked at the ability of melanoma cells to repair DNA damage by monitoring the histone protein H2A.X. Phophorylated H2A.X is rapidly expressed at sites of DNA damage, such as that caused by irradiation. With repair of the DNA, H2A.X is dephosporylated. We have a established a flow cytometry protocol where we irradiate the cells and look for the presence of H2A.X at 15 minutes after irradiation and 24 hours after irradiation. By measuring the decrease in phosphorylated H2A.X expression at 24 hours we can see how much repair has taken place in the cells. We have done this work in PC3 cells (a human prostate cell line) to act as a positive control and to perfect the technique. We have also done this in the non-drug exposed cell lines. We are double labeling the cells with propidium iodide to analyze cell cycle kinetics of the cells at the same time. To further validate the assay we are also using a fluorescent microscope to visualize H2A.X bound to a fluorescent tag and counting the number of cells positive at 15 minutes (when little repair will have taken place) and at 24 hours (when most repair will have occurred). As a control we are using the human prostate cancer cell line PC3 since it is known to express Cox-2. We have found that melanoma cells are able to avoid a G2/M cell cycle arrest that is normally found in irradiated cells, and which was found in the PC3 control cells. We have also found high H2A.X signal in both the control and melanoma cells 15 minutes after irradiation. The signal increases with increasing radiation dose as expected. The most interesting finding is that by 24 hours the H2A.X signal decreases in the melanoma cells indicating that they have repaired much of the radiation damage. This was not the case in the control PC3 cells. To confirm that these results were due to repair of radiation damage a split dose experiment was also done. Each of the cell lines were irradiated to 8Gy in one fraction or 8Gy in two 4Gy fractions 15 minutes, 1 hr or 6 Hrs apart. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
We did not find significant differences in repair in the Cox-2 exposed cells. Characterize the role of VEGF and its receptors in melanoma radiation resistance. We have measured VEGF using the Quantikine ELISA kit which is specific for canine VEGF. We have collected the samples from the cells and culture medium for each of the cell lines using 0Gy as a control and 2 & 8Gy. We have run the ELISA tests and found that Melanoma cells do produce VEGF. We also have also identified mRNA from the melanoma cells by PCR for VEGF and KDR and FLT-1 the melanoma receptors which may indicate an autocrine loop. To further investigate this we plan to test the effect of VEGF inhibition on cell proliferation. We have looked at Cox-2 inhibition and radiation resistance in canine melanoma and didn't find an effect. We developed an assay to measure repair of DNA in melanoma and found they can repair radiation-induced damage. We have also found that VEGF is expressed by melanoma cells in culture and are investigating the effects that its inhibition Western blot from Cell lines 12, 23 & 50 for VEGF using R&D systems canine VEGF antibody. We have also shown that melanoma cells express BCL-2 and that we can cause its own-regulation by the use of the drug Genasense. We have added the goal of measuring repair of canine melanoma since we are now able to measure this. The work to complete the VEGF section of the grant has been delayed as the inhibitor was no longer available. A commercially available canine VEGF antibody that can be used to block VEGF is now available from. We plan to use this to carry out the rest of the unfinished aims for this study and plan to carry this out over the next 6 months.
- No publications reported this period