Figure 1.
Fhit-deficient cells exhibit spontaneous DNA breaks.
(A) Neutral comet assays of HEK293 cells 2 days after transfections with siRNAs and pRcCMV-FHIT-flag or pRcCMV-empty-flag plasmids. Representative nuclei are shown; bars, 20 µm. (B) Box plots of Tail moments include data (siCtrl, n = 183; siFHIT, n = 142; siFHIT+CMV-ev, n = 135; siFHIT+CMV-Fhit, n = 132) from 3 separate experiments. Statistical significance was determined using the Kruskal-Wallis rank sum test. (C) Indirect immunofluorescence of γH2AX and 53BP1, 2 days after Fhit knockdown. Representative nuclei are shown; bars, 10 µm. (D and E) Quantification of γH2AX-positive cells (D) and 53BP1-positive cells (E). Bar graphs indicate the means, and error bars represent the standard deviations. Data were collected from 3 independent experiments. Statistical significance was assessed using a 2-sided Student's T-test. (F) Neutral comet assays of Fhit+/+ or Fhit−/− mouse kidney cells 48 h after transfection with pRcCMV-FHIT-flag or pRcCMV-empty-flag plasmids. Box plots of Tail moments are shown. Statistical significance was determined using the Mann-Whitney rank sum test. (G) Neutral comet assays of Fhit-deficient H1299 lung carcinomas cells, with or without induction of Fhit expression. Comet assays were performed 48 h after ponasterone A-induction of Fhit expression. Box plots of Tail moments are shown. Statistical significance was determined using the Mann-Whitney rank sum test.
Figure 2.
Loss of Fhit causes replication stress.
(A) Cyclin A and γH2AX indirect immunofluorescence after Fhit knockdown. Representative images are shown; bars, 10 µm. (B) Data obtained in (A) were quantified from 3 independent experiments, and statistical significance was determined using a 2-sided T-test. Bar graphs represent the means, and error bars mark the standard deviations. (C) pATR immunofluorescence 2 days after Fhit knockdown in HEK293 cells. Representative images are shown; bars, 5 µm. (D) Quantification of cells positive for more than 5 pATR foci/cell from 3 independent experiments; statistical significance was determined using a 2-sided T-test. (E) Neutral comet assays in siRNA transfected HEK293 cells treated with 2 mM hydroxyurea for 4 h. Box plots show quantification of Tail moments. P-values were determined using the Mann-Whitney rank sum test. (F) Neutral comet assays in H1299 E1 and D1 cells with ponasterone A-induction treated with 2 mM hydroxyurea for 4 h. Box plots show quantification of Tail moments. P-values were determined using the Mann-Whitney rank sum test.
Figure 3.
Loss of Fhit expression causes fork stalling.
(A) siRNA transfected HEK293 cells were pulse-labeled with CldU for 30 min, washed and pulse-labeled with IdU for 30 min. Representative indirect immunofluorescence images of labeled fibers are shown; bars, 10 µm. (B) Quantification of fork velocity in HEK293 cells. Fork velocity was determined by measuring lengths (µm) of IdU-labeled fibers and converting to kbp using a conversion factor of 1 µm = 2.59 kbp. Bars extending through boxplots indicate mean velocity, and bars contained within boxplots indicate median velocity. Statistical significance was determined using a 2-sided Student's T-test (n = 238 for siCtrl; n = 320 for siFHIT). (C) Representative images of sister forks proceeding outward from a common origin in siRNA transfected HEK293 cells; bars, 10 µm. DNA fibers from siRNA transfected HEK293 cells were prepared as in (A). (D) Scatter plots of distances traveled by left and right sister forks during pulse-labeling with IdU. The central area marked by red lines represents sister forks with less than 25% length difference. The percentages of asymmetric sister forks are indicated at the upper left region of plots. (E) Fork asymmetry is calculated as the ratio of the longest IdU tract to the shortest for each pair of sister forks. P-value was determined using the Mann-Whitney rank sum test. (F) DNA fiber analysis of fork velocity in Fhit+/+, Fhit−/− and Fhit−/− pRcCMV-FHIT-flag plasmid transfected mouse kidney cells. Quantification and statistical analysis was performed as described in (B). (G) DNA fiber analysis of fork velocity in H1299 E1 and D1 cells 48 h after ponasterone A treatment. Quantification and statistical analysis was performed as described in (B).
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.
Figure 5.
Fhit activates TK1 expression.
(A) Western blot analysis of TK1 and TYMS expression in siRNA transfected HEK293 cells. Western blots were performed on 5 independent experiments, and a representative blot is shown. (B) Western blot analysis of TK1 expression in siRNA transfected HEK293 cells with or with exogenous Fhit overexpression. pRcCMV expression plasmid carrying FHIT cDNA was co-transfected with FHIT siRNAs to achieve exogenous Fhit overexpression. A representative blot is shown. (C) Western blot analysis of TK1 expression in Fhit+/+ and Fhit−/− mouse kidney epithelial cells. A representative blot is shown. (D) Western blot analysis of TK1 expression in A549 cells with Fhit stably knocked down for 7–9 weeks. A representative blot is shown.
Figure 6.
Loss of Fhit causes replication stress-induced chromosomal instability.
(A) Cyclin A and 53BP1 immunofluorescence after Fhit knockdown in HEK293 cells. Representative images are shown; bars, 20 µm. (B and C) Histograms of 53BP1 nuclear bodies/G1 phase cell 3 days following siRNA transfection (B) or 14 days after siRNA transfection with fresh siRNAs transfected every 4 days (C). G1 phase cells were defined as cells negative for Cyclin A staining. Mann-Whitney rank sum test was used to determine P-values. (D) Representative images of DAPI-stained nuclei in siCtrl or siFHIT cells. Arrow marks a micronucleus; bars, 5 µm. (E) Quantification of micronucleated cells 3 days after siRNA transfections. Bar graphs represent the means, and error bars mark the standard deviations. P-value determined using a 2-sided T-test. (F) Percentage of aneuploid or tetraploid kidney cells established from Fhit+/+ or Fhit−/− mouse kidney epithelial cells. Cells were sub-cultured 8 times and metaphase chromosomes were prepared and counted (n = 37 for Fhit+/+; n = 40 for Fhit−/− metaphases). (G) Quantification of the number of breaks/metaphase for the mouse kidney cells described in (F).
Figure 7.
Genomic instability in Fhit-deficient cells correlates with onset of rapid proliferation and immortalization.
(A) Analysis of Fhit+/+ and Fhit−/− 3T3 MEF cell lines (n = 3, cell lines established from 3 embryos for each mouse strain). Arrows mark the passage numbers when MEFs became immortalized. (B) Western blot of Fhit+/+ MEFs for Fhit and GAPDH expression. Immunoblots were performed on lysates obtained at the indicated passage number. (C) Summary of copy number aberrations (CNAs) in pre- and post-senescence MEFs from Fhit+/+ and Fhit−/− mice. (D) The Fhit loss–induced genome instability model. Deletions in FHIT alleles occur due to FRA3B fragility causing loss of Fhit protein expression. Fhit loss causes dTTP pool insufficiency triggering replication stress, followed by stress-induced chromosomal instability. Chromosomal instability increases the likelihood of activating mutations in oncogenes and/or inactivating mutations in tumor suppressors, which are then selected for, facilitating cell transformation.