Fragile histidine triad (FHIT) gene deletion or promoter methylation and reduced Fhit protein expression occur in ∼70% of human epithelial tumors and, in some cancers, are clearly associated with tumor progression. Specific Fhit signal pathways have not been identified. We previously reported that compared with Fhit+/+ cells, Fhit−/− cells with an overactivated ATR/CHK1 pathway show increased mutation frequency and resistance to DNA damage–induced killing, indicating that Fhit and the CHK1 pathway have opposing roles in cells responding to DNA damage. In this study, we show that cells, with or without Fhit expression, have similar DNA double-strand break induction levels and similar rejoining rates following ionizing radiation, indicating that the effect of Fhit on cell radiosensitivity is independent of nonhomologous end-joining. By combining I-SceI–induced-DNA double-strand break system and small interfering RNA approach, we also show that knocking down Fhit increases the efficiency of homologous recombination repair of cells, but knocking down Chk1 decreases the efficiency of homologous recombination repair, associated with the sensitivity to ionizing radiation–induced killing. Taken together, the results show that the role of Fhit in affecting the sensitivity of cells to ionizing radiation–induced killing is through the CHK1 pathway linked to homologous recombination repair. These results also illustrate the importance of balanced checkpoint activation in genomic stability and suggest a connection between the radioresistance and mutagenesis, carcinogenesis, as well as tumor progression in Fhit-deficient cells or tissue.
The fragile histidine triad (FHIT) gene, encompassing the fragile site, FRA3B, at human chromosome 3p14.2 (1), has been reported to be absent in a number of human epithelial tumors, particularly in those tumors resulting from exposure to environmental carcinogens (2). Absence or reduction of FHIT gene expression has also been reported to be associated with a more aggressive progression of neoplasias (3, 4). However, the mechanism by which Fhit contributes to tumor progression has not been defined to date. We previously reported that compared with Fhit+/+ cells, Fhit−/− cells with an overactivated ATR/CHK1 pathway exhibit higher mutation frequency and with more resistance to multi–DNA damage inducers, including ionizing radiation (5, 6), indicating that Fhit and the ATR/CHK1 pathway have opposing effects on the radiosensitivity of cells. In response to DNA damage, proliferating cells slow their progress through the cell cycle by activating the DNA damage–induced checkpoints, which are believed to promote DNA repair (7–10). CHK1, as a substrate of ATR, is one of the most important signal transducers regulating the multiple checkpoints after DNA damage (11–14). CHK1 is involved in ionizing radiation–induced checkpoint regulation and protects mammalian cells from ionizing radiation–induced killing (13, 15–17), indicating the importance of the ATR/CHK1 pathway in maintaining cell survival following ionizing radiation. We showed that the ATR/CHK1 pathway protecting cells from ionizing radiation–induced killing is linked to homologous recombination repair, but independent of nonhomologous end-joining (NHEJ) repair (18, 19). It is clear that the normally activated checkpoint is important for keeping genetic integrity and stability. However, an overactivated checkpoint could promote overactivated homologous recombination repair and enhance the ability of cells associated with misrepair to tolerate DNA damage, resulting in an increase of both spontaneous and induced mutations. In this study, we show that the effect of Fhit on radiosensitivity is independent of NHEJ repair but is linked to homologous recombination repair. These results suggest that the increased survival of Fhit−/− cells following ionizing radiation is due to the fraction of cells with an increased mutation burden; cells would normally die if wild-type Fhit were expressed. Therefore, the radioresistance in cells with reduction or deficiency of Fhit is associated with carcinogenesis and tumor progression.
Materials and Methods
Cell lines and irradiation. Fhit+/+ and Fhit−/− epithelial cells from mouse kidney, generated as described earlier (20), were immortalized by tissue culture passaging. These cells were adapted to growth in DMEM supplemented with 10% iron-supplemented calf serum (Sigma-Aldrich, Co., St. Louis, MO) at 37°C in an atmosphere of 5% CO2 and 95% air. Irradiation of cells was done by exposing cells to X-rays (310 kV, 10 mA, 2 mm Al filter).
Induction and rejoining of DNA double-strand breaks. Analysis of induction and rejoining of DNA double-strand breaks is done by using the asymmetrical field inversion gel electrophoresis (FIGE) assay as described before (18). Briefly, cells in cold medium were irradiated and returned to the incubator at 37°C. At various times thereafter, cells were collected and mixed with an equal volume of 1% agarose (InCert agarose, FMC Bioproducts, Rockland, ME). A similar protocol was also used to determine induction of DNA double-strand breaks, except that, in this case, cells were embedded in agarose blocks before irradiation and were placed in lysis buffer [10 mmol/L Tris (pH 8.0), 50 mmol/L NaCl, 0.5 mol/L EDTA, 2% N-lauryl sarcosyl, 0.1 mg/mL proteinase E] immediately after irradiation. Blocks in lysis buffer were incubated first at 4°C for 45 minutes, and then at 50°C for 16 to 18 hours. The blocks were washed in a buffer containing 10 mmol/L Tris (pH 8.0) and 0.1 mol/L EDTA, and treated at 37°C for 1 hour with 0.1 mg/mL RNase A in the same buffer. Asymmetrical FIGE was carried out in 0.5% Seakem agarose (FMC) in 0.5× Tris-borate EDTA [45 mmol/L Tris (pH 8.2), 45 mmol/L boric acid, 1 mmol/L EDTA] at 10°C for 40 hours. During this time, cycles of 1.25 V/cm for 900 seconds in the direction of DNA migration alternated with 5 V/cm for 75 seconds in the reverse direction. The agarose gels were stained with ethidium bromide (5 μg/mL) for 6 hours at room temperature and washed with water for 1 hour. DNA double-strand breaks were quantitated by calculating the fraction of activity released from the well into the lane in irradiated and nonirradiated samples by means of a fluorescence image measured with a PhosphorImager (Typhoon 8600, Molecular Dynamics, Piscataway, NJ).
Fhit and Chk1 small interfering RNA design and transfection. Fhit small interfering (siRNA) that specifically targets the sequences of the mouse Fhit mRNA (5′-AAGCAUUUCCAGGGGACCUCC-3′) was designed. The designed siRNA was synthesized by Dharmacon Company (Lafayette, CO). The Scrambled Duplex RNA (D-1200-5, Dharmacon Company) was used as the control RNA. Chk1 siRNA was as described before (6). The siRNA transfections were done with OligofectAMINE (Invitrogen, Carlsbad, CA) following the instructions of the manufacturer. At 36 hours following transfection, the cells were collected for further examination (homologous recombination repair, Western blot, and clonogenic assay). Western blot analysis of protein levels was done by whole cell lysis; briefly, 5 × 105 cells were lysed in 25 μL of radioimmunoprecipitation assay lysis buffer [50 mmol/L Tris-HCl (pH 7.4); 1% NP40; 0.25% sodium deoxycholate; 150 mmol/L NaCl; 1 mmol/L EGTA; 1 mmol/L phenylmethylsulfonyl fluoride; 1 μg/mL each of aprotinin, leupeptin, and pepstatin; 1 mmol/L Na3VO4; 1 mmol/L NaF] and mixed with 25 μL of 2× protein loading buffer. Antisera against Fhit (20), CHK1 (sc-8404, Santa Cruz Biotechnology, Inc.), and proliferating cell nuclear antigen (PCNA, sc-56, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used in Western blot analyses.
Homologous recombination repair assay. The homologous recombination repair assay was done using the pDR-GFP system (obtained from Dr. Jasin's laboratory; ref. 21). Fhit wild-type cells were transfected with pDR-GFP plasmid containing a mutated GFP gene with an 18 bp I-SceI site. The stably transfected cell lines were selected by growing in medium containing 5 μg/mL of puromycin. Puromycin-resistant colonies were screened by Southern blots for intact DR-GFP reporter genomes. The positive cell lines were designated F-DRGFP. To evaluate homologous recombination repair of DNA double-strand breaks, F-DRGFP cells were either transfected with pGFP (containing full-length GFP cDNA) or transfected with pCMV3xnlsI-SceI plasmid (containing full-length I-SceI expression sequences, obtained from Dr. Nickoloff's laboratory; ref. 22). Transient expression of I-SceI endonuclease generates a double-strand break at the integrated GFP gene sequences and stimulates homologous recombination repair. GFP signal was assayed at 2 days posttransfection by flow cytometry (Beckman Coulter, Fullerton, CA, XL/MCL). Results were collected as dot plots of PMT1 (525 bp) to facilitate distinction between GFP-positive and GFP-negative cells. The frequency of recombination events was calculated from the frequency of GFP signal in F-DRGFP cells transfected with I-SceI by subtracting the frequency of GFP signal in F-DRGFP cells without transfection and dividing by the frequency of GFP signal in F-DRGFP cells transfected with pGFP.
Clonogenic cell survival. The sensitivity of the F-DRGFP cells to ionizing radiation–induced killing was examined using clonogenic cell survival assay previously described (6). Briefly, the F-DRGFP cells were transfected with either Fhit or Chk1 siRNA. Thirty-six hours later, the cells were exposed to X-ray (4 Gy). The cells were then collected and plated, aiming at 20 to 200 colonies per dish (60 mm). Two replicates were prepared for each datum point and were incubated for 1 week to allow colonies to develop. Colonies were stained with crystal violet (100% methanol solution) before counting.
Results and Discussion
Fhit+/+ and Fhit−/− cells show similar DNA double-strand break induction levels and similar double-strand break rejoining rates following ionizing radiation. Ionizing radiation–induced DNA double-strand breaks are the most severe damage affecting cell survival. NHEJ and homologous recombination repair are the two major pathways for repairing DNA double-strand breaks in mammalian cells. Compared with Fhit+/+ cells, Fhit−/− cells are resistant to ionizing radiation–induced killing (6), indicating that Fhit is involved in DNA repair either directly or indirectly. However, clarification is needed for the pathway by which Fhit affects cell radiosensitivity, through NHEJ or homologous recombination repair, or both. The ATR/CHK1 pathway affecting the radiosensitivity of cells involves homologous recombination repair but is independent of NHEJ (18, 23) and, without Fhit, the cells exhibit an overactivated ATR/CHK1 pathway (6). Therefore, the effect of Fhit on cellular radiosensitivity might be expected to be independent of NHEJ and linked to homologous recombination repair. To test this hypothesis, we compared the NHEJ abilities of Fhit+/+ cells and Fhit−/− cells by using an asymmetrical FIGE assay. The results show that cells with or without Fhit have similar double-strand break induction levels and similar rejoining rates of DNA double-strand breaks following ionizing radiation (Fig. 1), although they have different sensitivities to ionizing radiation–induced killing (6). These results indicate that the effect of Fhit on cell radiosensitivity is not through affecting NHEJ repair, but might be linked to homologous recombination repair.
Fhit and Chk1 small interfering RNAs have opposing effects on homologous recombination repair efficiency. To study the effect of Fhit on homologous recombination repair efficiency following DNA double-strand breaks, we combined the pDR-GFP system (obtained from Dr. Jasin's laboratory; ref. 21) and siRNA approaches. The Fhit+/+ cells were transfected with pDR-GFP plasmid containing a mutated GFP gene with an 18 bp I-SceI site. Stably transfected cell lines were selected by growing them in medium containing 5 μg/mL of puromycin. Puromycin-resistant colonies were screened by Southern blot for an intact DR-GFP reporter (Fig. 2A). Positive clones were designated F-DRGFP and ∼20% of puromycin-resistant clones had integrated an intact DR-GFP repair substrate. To evaluate double-strand break homologous recombination repair, F-DRGFP cells were either transfected with pGFP (containing full-length cDNA of GFP) or transfected with pCMV3xnlsI-SceI plasmid (containing full-length I-SceI expression sequences). Twenty-four hours later, the cells were transfected with either Fhit siRNA or Chk1 siRNA. The cells were collected for measuring the GFP signal at 36 hours after siRNA transfection. The results show that the siRNAs specifically inhibit the respective protein expression (Fig. 3A). When Fhit expression was inhibited in F-DRGFP-clone3 (F-3) or F-DRGFP-clone6 (F-6) cells, the efficiencies of homologous recombination repair in these cells are much higher than in the non–RNA-treated control cells. By contrast, when CHK1 expression was inhibited (Fig. 2B and C), the efficiencies of homologous recombination repair in these cells are much lower than in the non–RNA-treated control cells. These results clearly indicate that Fhit and CHK1 have opposing effects on homologous recombination repair: CHK1 promotes homologous recombination repair and Fhit inhibits homologous recombination repair. It is believed that Fhit affects homologous recombination repair by affecting the ATR/CHK1 pathway because without Fhit, the cells show an overactivated ATR/CHK1 pathway and resistance to DNA damage–induced cell killing (5, 6).
Fhit and Chk1 small interfering RNAs have opposing effects on cellular sensitivity to ionizing radiation–induced killing. To further study whether the effects of Fhit and Chk1 siRNAs on homologous recombination repair are linked to the cell sensitivity to ionizing radiation–induced killing, we examined the clonogenicity in the F-DRGFP cells treated with either Fhit or Chk1 siRNA following ionizing radiation. The results show that when the level of Fhit protein is reduced by Fhit siRNA treatment (Fig. 3A), both F-3 and F-6 cells show increased resistance to ionizing radiation–induced killing (Fig. 3B), but when the level of CHK1 protein is reduced by Chk1 siRNA treatment (Fig. 3A), both F-3 and F-6 cells show increased sensitivity to ionizing radiation–induced killing (Fig. 3B), indicating a functional connection between radiosensitivity and homologous recombination repair: The higher the efficiency of homologous recombination repair, the more radioresistance is shown by the cells. Previously, we reported that the effect of CHK1 on radiosensitivity is independent of NHEJ (18, 19) and that Fhit affecting the sensitivity of cells to DNA damage inducers is through affecting the ATR/CHK1 pathway (5, 6). Therefore, we believe that effects of both Fhit and CHK1 on the sensitivity to ionizing radiation–induced killing are through effects on homologous recombination repair and are independent of NHEJ. The mechanism by which Fhit affects the ATR/CHK1 pathway following DNA damage needs future elucidation. Homologous recombination repair is thought to be an error-free repair compared with NHEJ. However, overexpressing Rad51, an essential factor for homologous recombination repair, results in more mutations in the cells (24). Ku deficiency shows the overactivated homologous recombination repair pathway (25, 26) and is also associated with more neoplastic growth (27). These results suggest that overactivated homologous recombination repair does not help cells to maintain genetic stability over the long term. Fhit−/− cells show a higher mutation frequency in response to DNA damage inducers (5), which is reasoned that the increased survival of Fhit-deficient cells following ionizing radiation is related to carcinogenesis and tumor progression through enhanced survival of cells carrying an increased mutation burden.
Note: B. Hu and H. Wang contributed equally to this work.
Grant support: Department of Defense grant BC023350 (Y. Wang), National Aeronautics and Space Administration grant NAG2-1628 (Y. Wang), and CA77738 (K. Huebner).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Drs. M. Jasin (Memorial-Sloan Kettering, Department of Molecular Biology, New York, NY) and J.A. Nickoloff (University of New Mexico HSC, Department of Molecular Genetics and Microbiology, Albuquerque, NM) for reagents and Nancy Mott for help in the preparation of the manuscript.