Figure 4. Fhit modulates dTTP pools to prevent DNA breaks.

(A) Deoxyribonucleotide triphosphate (dNTP) levels in HEK293 cells 72 h after Fhit knockdown. Bar graphs represent means of 4 independent experiments, and error bars denote the standard deviations. The P-values were determined using a 2-sided T test; ns = not significant. (B) Correlation of relative Fhit expression and relative dTTP levels. siRNA transfected HEK293 cells were split into matching pairs, one for dNTP analysis and the other for western blot analysis of Fhit knockdown. Relative Fhit expression in Fhit knockdown cells compared to control cells was determined by densitometry and normalized to GAPDH expression. Relative dTTP levels were defined as dTTP concentration in siFHIT cells/dTTP concentration in siCtrl cells. (C) dNTP measurements in A549 cells with 7–9 week stable Fhit knockdown. Bar graphs represent means of 7 independent experiments, and error bars denote the standard deviation. P-values were calculated as in (A). (D) Box plots of Tail moments measured from neutral comet assays of HEK293 cells with Fhit knockdown, untreated or supplemented daily with 10 µM thymidine for 48 h (siCtrl mock, n = 242; siCtrl+thymidine, n = 156; siFHIT mock, n = 193; siFHIT+thymidine, n = 115). Statistical significance was determined using the Kruskal-Wallis rank sum test. (E) DNA fiber analysis of fork velocity in siRNA transfected HEK293 cells supplemented daily with 10 µM thymidine for 48 h. Statistical significance was determined using a 2-sided Student's T-test (siCtrl mock, n = 136; siCtrl+thymidine, n = 152; siFHIT mock, n = 155; siFHIT+thymidine, n = 153). (F) DNA fiber analysis of sister fork asymmetry in siRNA transfected HEK293 cells supplemented daily with 10 µM thymidine for 48 h. Fork asymmetry and P-values were determined as in Figure 3E (siCtrl mock, n = 87; siCtrl+thymidine, n = 86; siFHIT mock, n = 96; siFHIT+thymidine, n = 93). dT = thymidine 10 µM; ns = not significantly different.