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Results: 4

1.
Figure 1

Figure 1. FdUrd mediated inactivation of TS versus TS shRNA mediated suppression of TS. From: shRNA suppression of thymidylate synthase produces DNA mismatches and results in excellent radiosensitization.

Whole cell lysates from HCT116 and HT29 cells were analyzed by Western blotting for TS expression. (A) HCT116 and (B) HT29 cells were incubated with an IC50 for FdUrd (24, 48, and 72 h), left untreated (represented by the control lane in blots describing TS expression following FdUrd), or transduced with either shRNA targeted to TS mRNA (TS1, and TS2) for 1–8 days, or with non-specific shRNA (represented by the control lane in blots describing TS expression following shRNA transduction). A representative blot from a minimum of three separate experiments is shown.

Sheryl A. Flanagan, et al. Int J Radiat Oncol Biol Phys. 2012 December 1;84(5):e613-e620.
2.
Figure 3

Figure 3. TS shRNA-mediated suppression of TS is as effective as inactivation of TS by FdUrd as a radiosensitizing strategy. From: shRNA suppression of thymidylate synthase produces DNA mismatches and results in excellent radiosensitization.

The effect of FdUrd and TS shRNA on the sensitivity of (A) HCT116 and (B) HT29 cells to ionizing radiation compared to untreated (control) cells was examined by incubation of cells with an IC50 for FdUrd (24 h), or transduction with shRNA (5 d) targeted to TS mRNA (TS1, and TS2) or non-specific (NS) shRNA, followed by radiation. The surviving fraction was corrected for cell death attributable to FdUrd, TS shRNA or NS shRNA alone, respectively. Data shown are from a representative experiment. Each experiment was repeated at least three times in triplicate.

Sheryl A. Flanagan, et al. Int J Radiat Oncol Biol Phys. 2012 December 1;84(5):e613-e620.
3.
Figure 2

Figure 2. Both TS shRNA mediated suppression of TS and FdUrd mediated inactivation of TS elicit a decrease in dTTP. From: shRNA suppression of thymidylate synthase produces DNA mismatches and results in excellent radiosensitization.

dNTPs were extracted from untreated (control) (A) HCT116 and (B) HT29 cells and following incubation with an IC50 for FdUrd (4, 24, 48, and 72 hr), or transduction with shRNA targeted to TS mRNA (TS1, and TS2) for 2, 5, and 8 days, or with non-specific (NS) shRNA. The data are presented as a % of the corresponding control value and represent the mean ± SD from duplicate determinations. Control values (pmol dNTP/106 cells): HCT116 0-1 cells, dCTP: 8.5 ± 3.0, dTTP: 60 ± 17.6, dATP: 8.8 ± 0.7, dGTP: 3.6 ± 0.7. HT29 cells, dCTP: 17.8 ± 10.0, dTTP: 92 ± 33.0, dATP: 20 ± 7.8, dGTP: 5.3 ± 3.5.

Sheryl A. Flanagan, et al. Int J Radiat Oncol Biol Phys. 2012 December 1;84(5):e613-e620.
4.
Figure 4

Figure 4. FdUrd mediated inactivation of TS or TS shRNA mediated suppression of TS produces an increase in DNA mismatches. From: shRNA suppression of thymidylate synthase produces DNA mismatches and results in excellent radiosensitization.

pSP189 plasmid mutation frequency was determined in (A) HCT116 and (B) HT29 . The mutation frequencies in plasmids replicated in the untreated control group of both cell lines were similar 0.09 % ± 0.02, and 0.07 ± 0.03 %; HT29 or HCT116 cells, respectively; p=0.61). Statistical significance was determined by the application of a one tailed t -test. * significantly different from 1.0 (control), P<0.05. A one-way ANOVA followed by post tests was used to test for differences between treatments. Bars marked with * are not significantly different from each other, p<0.05. Total number of colonies counted: HCT116, Control = 44,128 FdUrd = 30,829, TS1 = 22,468, TS2 = 19,076; HT29, Control = 20,444, FdUrd = 16,287 , TS1 = 18.656, TS2 = 19,762.

Sheryl A. Flanagan, et al. Int J Radiat Oncol Biol Phys. 2012 December 1;84(5):e613-e620.

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