In response to genomic stress, proliferating cells slow their progress through the cell cycle by activating the DNA damage-induced checkpoints, G1, S, and G2-phase checkpoints, which are believed to promote DNA repair and to benefit genomic integrity (Paulovich and Hartwell, 1995; Eller et al., 1996; Paulovich et al., 1997; Zhou and Elledge, 2000). ATR is one of the most important signal transducers regulating the multiple checkpoints after DNA damage (Abraham, 2001). Expression of a dominant-negative ATR sensitizes mammalian cells to many different types of DNA damage and diminishes the ionizing radiation (IR)-induced G2/M checkpoint (Cliby et al., 1998; Wright et al., 1998), emphasizing the important roles of ATR in IR-induced checkpoint activation. The main downstream substrate of ATR for regulating the checkpoints is CHK1 (Martinho et al., 1998; Guo et al., 2000; Liu et al., 2000; Zhao and Piwnica-Worms, 2001). CHK1 is involved in IR-induced S and G2 checkpoints in mammalian cells (Liu et al., 2000; Zhao et al., 2002; Zhou et al., 2002). Blocking the ATR/CHK1 pathway abolishes the checkpoints regulated by this pathway and sensitizes the cells to IR-induced killing (Hu et al., 2001; Wang et al., 2002b), suggesting the importance of the checkpoints in maintaining cell radiosensitivity. However, over-activated checkpoint response could enhance the ability of cells to tolerate DNA damage when their genomes contain un-repaired or mis-repaired damage, which results in an increase of both spontaneous and induced mutations.
The Fragile Histidine Triad (Fhit) gene, encompassing the fragile site, FRA3B, at human chromosome 3p14.2 (Ohta et al., 1996) has been reported to be deleted in a number of human epithelial tumors, particularly in those tumors resulting from exposure to environmental carcinogens (Croce et al., 1999). Absence or reduction of Fhit gene expression has also been reported to be associated with a more aggressive progression of neoplasias (Gatalica et al., 2000; Huebner and Croce, 2003) although specific pathways through which Fhit contributes to tumor progression have not been defined. In our initial studies of responses of Fhit deficient cells to various stressful stimuli, we found that Fhit deficient cells are significantly resistant to UVC with higher mutation rates (Ottey et al., 2004). We report here that Fhit−/− cells derived from mice (Fong et al., 2000; Turner et al., 2002) have stronger IR-induced S and G2 checkpoint responses than Fhit+/+ cells. An over-activated ATR/CHK1 pathway regulates the stronger checkpoint responses shown in Fhit−/− cells, which is responsible for the radioresistance of Fhit−/− cells. These results suggest a model to explain the association between Fhit deficiency and tumor progression.
MATERIALS AND METHODS
Cell lines, chemical treatment, and irradiation
Fhit+/+ and Fhit−/− epithelial cells from mouse kidney, generated as described earlier (Fong et al., 2000), were immortalized by tissue culture passaging. These cells were adapted to growth in DMEM supplemented with 10% iron-supplemented calf serum (Sigma-Aldrich Co., USA) at 37°C in an atmosphere of 5% CO2 and 95% air. Caffeine (Sigma-Aldrich Co.), or UCN-01 (NCI, USA) was added to the culture 30 min before the cells were exposed to X-rays (310 kV, 10 mA, 2-mm Al filter) and was kept in the culture until the cells were collected.
The S phase (S) checkpoint is detected by measuring DNA synthesis, which is similar to that described previously (Zhou et al., 2002). Briefly, 1 × 105 cells from a growing culture were seeded in 60-mm tissue culture dishes with 3 ml of medium containing 10 nCi of [14C]-thymidine and allowed to grow for more than one doubling time. This [14C] pre-labeling provides an internal control for cell number by allowing normalization for total DNA content of samples.
Before irradiation, the cell cultures were changed with pre-warmed medium (washed off [14C]-thymidine) containing either caffeine or UCN-01 for 30 min. Cells were exposed to X-rays (310 kV, 10 mA, 2-mm Al filter) at room temperature and returned to 37°C. The chemicals were kept in the culture until cells were harvested. [3H]-thymidine at 0.5 μCi was added to the culture for 30 min at 3 h after IR and the cells were then collected. The rate of DNA synthesis for each sample was calculated as 3H dpm/14C dpm and is presented as a percentage of the control values obtained from sham-irradiated cells at the same time-point, as described previously (Zhou et al., 2002).
Flow cytometry assay
The G2 checkpoint is detected by flow cytometry measurement. As described (Hu et al., 2001), Fhit cells were collected at required times and fixed in 70% ethanol. Cells were washed with PBS and stained with a solution containing 62 μg/ml RNase A, 40 μg/ml propidium iodide, and 0.1% Triton X-100 in phosphate-buffered saline at room temperature for 1 h. The distribution of cells in the cell cycle was measured in a flow cytometer (Coulter Epics Elite, USA).
Kinase activity and Western blot
Nuclear extracts were prepared by using the NE-PER™ kit (PIERCE, USA) according to the manufacturer's instructions. The fractions of chromatin-bound extract were prepared as described previously (Wang et al., 2002a). Briefly, cells were collected and washed in cold phosphate-buffered saline. Proteins were then extracted with cold 0.1% Triton X-100 in CSK buffer (10 mM PIPES pH 6.8, 100 mM NaCl, 300 mM sucrose, 1 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfony fluoride) for 20 min at 4°C. The chromatin-bound fraction was then pelleted by low-speed centrifugation at 3,000 rpm for 5 min at 4°C. The supernatant was named fraction 1. These pellets were then re-extracted by incubating in the same CSK buffer and were collected by centrifugation at 3,000 rpm for 10 min at 4°C. This supernatant was named fraction 2. The final pellet fraction (containing chromatin-bound proteins) was solubilized in radioimmunopreciptation assay (RIPA) buffer (150 mM NaCl, 40 mM MOPS, pH 7.2, 1 mM EDTA, 1% NP40, 1% Sodium deoxycholate, 0.1% SDS) and was named fraction 3. For ATR kinase assay, 500 μg of fraction 3 was then mixed with 2 μg of ATR antibody (sc-1887, Santa Cruz Biotechnology, Inc., USA) in the presence of 20 μl of a 50% (v/v) protein G-Sepharose slurry (RepliGen, USA) in 500 μl of Buffer A (0.5% NP-40, 1 mM Na3VO4, 5 mM NaF, 0.2 mM PMSF in PBS buffer). For CHK1 kinase assay, 250 μg of nuclear extract was then mixed with 1 μg of CHK1 antibody (sc-7898, Santa Cruz Biotechnology, Inc.) in the presence of 10 μl of a 50% (v/v) protein A-Sepharose slurry in 250 μl of Buffer A. These mixtures were gently rotated overnight at 4°C. Immune complexes were washed twice with Buffer A, then twice with Buffer B (10 mM HEPES, pH 8.0, 50 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT). The ATR kinase immunoprecipitate supplemented with 1 μg of PHAS-1 (Stratagene, USA) and the CHK1 kinase immunoprecipitate supplemented with 1 μg of purified GST-CDC25C200–256 (Wang et al., 2002a) were incubated at 30°C for 30 min in 20 μl Buffer B containing 10 μCi (γ-32P) ATP. Samples were analyzed by 12% SDS–PAGE and the kinase activities determined by measuring the incorporation of 32P into PHAS-1 protein (ATR kinase) or into CDC25C200–256 (CHK1 kinase) with the PhosphoImager. Antibodies against ATR (sc-1887, Santa Cruz Biotechnology, Inc.), CHK1 (sc-8404, Santa Cruz Biotechnology, Inc.), and CDC25A (sc-7389, Santa Cruz Biotechnology, Inc.) were used in the Western Blot.
Cellular sensitivity to radiation was determined by the loss of colony-forming ability as described previously (Hu et al., 2001).
Transfection of Atr and Chk1 siRNA
The Atr siRNA was designed to specifically target the sequences of the conserved region between rat and mouse Atr mRNA (5′-AAGACAGATTCTCTGCCAGTT-3′). The Chk1 siRNA was designed to specifically target the sequences of the conserved region among human, rat, and mouse Chk1 mRNA (5′-AAGTTCAACTTGCTGTGAATA-3′). The siRNAs were synthesized by Dharmacon, Inc., USA. Scrambled duplex RNAs (Dharmacon, Inc.) were used in the control transfection. The RNAs were delivered to the cells by OLIGOFECTAMINE™ (Invitrogen Corp., USA), according to the manufacturer's instructions. The cells were analyzed at 36 h posttransfection.
Stronger S checkpoint response shown in Fhit−/− cells
The IR-induced S checkpoint response is typically quantitated as a transient decrease in [3H]-thymidine incorporation after irradiation (Painter and Young, 1980; Xu et al., 2001). Recent results from our laboratory suggest the operation of two distinct but complementary pathways in the regulation of the S-phase checkpoint that rely on the ATM and ATR kinases, respectively, in mammalian cells (following IR) (Zhou et al., 2002). The ATM-dependent pathway is the fast response (immediately after IR and is maintained ∼2 h) and the ATR-dependent pathway is the slow response (∼1 h after IR and is maintained up to 6 h). To determine which of these pathways is involved in the response of the Fhit−/− cells, we examined these Fhit−/− cells for the inhibition of DNA synthesis at different times following IR. There is not much difference in the inhibition of DNA synthesis between Fhit+/+ and Fhit−/− cells at 0.5 h after IR (data not shown). There is a stronger inhibition of DNA synthesis in Fhit−/− cells than that seen in Fhit+/+ cells at 3 h after IR (Fig. 1A), suggesting a stronger slow S checkpoint response in the absence of Fhit protein. The slow S checkpoint response in irradiated cells is ATM-independent and ATR/CHK1-dependent (Zhou et al., 2002). To test whether this was the affected pathway in Fhit−/− cells, we examined the effects of caffeine (non-specific inhibitor of ATR) or UCN-01 (non-specific inhibitor of CHK1) on the inhibition of DNA synthesis in these Fhit cells at 3 h after IR. The results are shown in Figure 1. Caffeine or UCN-01 abolishes the stronger inhibition of DNA synthesis in Fhit−/− cells, suggesting that an over-activated ATR/CHK1 pathway exists in irradiated Fhit−/− cells.
Stronger G2 checkpoint response shown in Fhit−/− cells
There are two molecularly distinct G2/M checkpoint responses in mammalian cells following IR (Xu et al., 2002). The fast response representing a mitosis accumulation of cells that were in G2 phase at the time of IR is ATM-dependent and the slow response representing G2 accumulation of cells that were in earlier phases at the time of IR is ATM-independent (Xu et al., 2002) but ATR/CHK1 dependent (Hu et al., 2001; Wang et al., 2002b). If there was an over-activated ATR/CHK1 pathway in irradiated Fhit−/− cells, as described above, the irradiated Fhit−/− cells should show an enhanced G2 accumulation. To test this hypothesis, we examined the G2 accumulation in these irradiated Fhit cells. The results showed that without IR, Fhit−/− cells had more S phase cells than Fhit+/+ cells (Fig. 1B) although these two cell lines had a similar doubling time, suggesting an active S checkpoint in non-irradiated control cells. After IR (4 Gy), G2 accumulation occurs earlier and the level of the G2 accumulation is higher in irradiated Fhit−/− cells than that in Fhit+/+ cells (Fig. 1B), indicating a stronger G2 checkpoint in irradiated Fhit−/− cells. These data provide additional evidence to support the hypothesis that there is an over-activated ATR/CHK1 pathway in irradiated Fhit−/− cells.
An over-activated ATR/CHK1 pathway exists in the irradiated Fhit−/− cells
To further study the hypothesis that there is an over-activated ATR/CHK1 pathway in irradiated Fhit−/− cells, we measured ATR and CHK1 kinases activities in the Fhit cells. No difference was noted in ATR activity between irradiated and control samples from both cytoplasmic and nuclear extracts (data not shown). ATR activity of the chromatin-bound fraction, however, was higher in irradiated (6 h) than non-irradiated cells for both cell lines (Fig. 2) suggesting that this pool of ATR contains the protein activated in response to DNA damage. A higher level of ATR activity was shown in irradiated Fhit−/− cells than in irradiated wild-type cells (Fig. 2), indicating an over-activated ATR in irradiated Fhit−/− cells. The main downstream target of ATR for regulating checkpoints is CHK1 (Martinho et al., 1998; Guo et al., 2000; Hekmat-Nejad et al., 2000; Liu et al., 2000; Deming et al., 2001; Lopez-Girona et al., 2001; Zhao and Piwnica-Worms, 2001). By using whole cell extracts, we observed a stronger phosphorylated CHK1 signal in Fhit−/− cells than in Fhit+/+ cells (Fig. 2A), confirming the over-activated ATR in irradiated Fhit−/− cells. In addition, we observed higher CHK1 kinase activity in Fhit−/− cells than in Fhit+/+ cells following IR, suggesting the over-activated CHK1 in irradiated Fhit−/− cells (Fig. 2A).
The CHK1-regulated checkpoint is mediated by an inhibition of the CDC25A phosphatase (Zhao et al., 2002) that activates the CDC2 or CDK2 kinase by removing inhibitory phosphates (Thr14 and Tyr15). CHK1 could phosphorylate CDC25A, thus resulting in degradation of this phosphatase. To examine whether activation of the CHK1 kinase was associated with degradation of CDC25A, we measured CDC25A levels in these Fhit cells. A lower level of CDC25A was shown in Fhit−/− cells than in Fhit+/+ cells following IR (6 h) (Fig. 2A), consistently with ATR and CHK1 activation results (Fig. 2). Altogether the results indicate that an over-activated ATR/CHK1 pathway exists in irradiated Fhit−/− cells, suggesting that the over-activated ATR/CHK1 pathway is responsible for the stronger checkpoint responses in such cells.
Atr and Chk1 siRNAs abolish the stronger checkpoint responses in irradiated Fhit−/− cells
To confirm that the ATR/CHK1 pathway is responsible for the stronger checkpoint responses in irradiated Fhit−/− cells, we examined the effects of Atr and Chk1 siRNAs on the checkpoint responses. Atr and Chk1 siRNAs specifically inhibited expressions of ATR and CHK1 in the transfected cells (Fig. 3A) and abolished both the stronger S checkpoint (Fig. 3B) and the stronger G2 checkpoint (Fig. 3C) in the irradiated Fhit−/− cells. These results provide direct evidence that the ATR/CHK1 pathway plays a key role in the stronger checkpoint responses in irradiated Fhit−/− cells.
It should be mentioned that the Chk1 siRNA used in this study is designed to target the sequences at 65–85 nucleotides from the start codon region, the conserved region among human, rat, and mouse Chk1 mRNA (5′-AAGTTCAACTTGCTGTGAATA-3′). Although it is usually suggested that siRNA should be designed at least 75–100 nucleotides downstream of the start codon of the target mRNA, this Chk1 siRNA works well not only on mouse cells (Fig. 3A), but also on human and rat cells (data not shown).
Fhit−/− cells are more resistant to IR-induced killing than Fhit+/+ cells
S and G2 checkpoints facilitate homologous recombination repair (HRR) and, therefore, affect the radioresistance of cells (Wang et al., 2003a,b). The stronger S and G2 checkpoints regulated by the over-activated ATR/CHK1 pathway exist in irradiated Fhit−/− cells, suggesting that Fhit−/− cells are more radioresistant than Fhit+/+ cells. To test this hypothesis, we measured the clonogenic survival of Fhit+/+ and Fhit−/− cells following IR. As expected, Fhit−/− cells were more resistant to IR-induced killing than Fhit+/+ cells (Fig. 4A).
To examine whether the over-activated ATR/CHK1 pathway that regulated the stronger S and G2 checkpoints caused the radioresistance of Fhit−/− cells, we examined the effects of Atr and Chk1 siRNAs on the radiosensitivity of these Fhit cells. Atr and Chk1 siRNAs radiosensitized both Fhit+/+ and Fhit−/− cells, but the sensitization in Fhit−/− cells is larger than that in Fhit+/+ cells (Fig. 4B), resulting in similar radiosensitivities of Fhit+/+ and Fhit−/− cells. These results suggest that the radioresistant phenotype of Fhit−/− cells depends on the stronger checkpoint response.
The results of this study indicate for the first time that Fhit−/− cells show stronger ATR/CHK1-regulated S and G2 checkpoint responses, which contributes to the radioresistance of these cells. To confirm that the over-activated ATR/CHK1 pathway in irradiated Fhit−/− mouse cells is related to the absence of Fhit, we carried out similar experiments in one pair of Human Fhit+/+ and Fhit−/− cells (established in Dr. Huebner's laboratory). Similar to mouse cell lines, the human Fhit+/+ cells with a higher ratio of S phase cells have a similar doubling time with the human Fhit−/− cells (Ottey et al., 2004). After IR, the human Fhit−/− cells also show the stronger checkpoint response than their counterparts (data not shown), indicating that the over-activated ATR/CHK1 pathway in irradiated Fhit−/− mouse cells is because of the absence of Fhit.
DNA DSB is the most severe damage for cell survival induced by IR. Two major complementary DSB repair pathways exist in eukaryotic cells, homologues recombination repair (HRR) and non-homologous end joining (NHEJ). HRR is the major pathway to repair DSB in yeast and NHEJ was originally thought to be the only major DSB repair process in mammalian cells. Recently, HRR was also found to be a major DNA DSB repair process in addition to NHEJ in mammalian cells (Liang et al., 1998; Pastink et al., 2001; Thompson and Schild, 2001). Although Fhit−/− cells are resistant to IR-induced killing, they have a normal rejoining ability of DNA DSBs that resulted from using asymmetric field inversion gel electrophoresis (AFIGE) (our unpublished data), suggesting that the radioresistance shown in Fhit−/− cells is independent of NHEJ. We previously reported that NHEJ is a process independent of checkpoint and that HRR mainly benefits from checkpoint activation (Wang et al., 2003a,b). The radioresistance of Fhit−/− cells might be caused by higher HRR, which is benefited by the stronger checkpoint responses. In addition to DNA repair, the other factor affecting irradiated cells survival is the ratio of apoptosis although apoptosis is not the major factor to affect the DNA-damaged cell death in solid tumors (Brown and Wouters, 1999). With stronger checkpoint responses, Fhit−/− cells showed less apoptosis cells than Fhit−/− cells following UV (Ottey et al., 2004), suggesting that checkpoints, particular S and G2 checkpoints, protect cells from apoptosis. Stronger checkpoints facilitate HRR and, therefore, reduce the cells apoptosis. However, this hypothesis needs to be tested.
Absence or reduction of Fhit gene expression occurs in a number of human epithelial cancers resulting from exposure to environmental carcinogens and is associated with progression to more aggressive neoplasias, but the mechanism remains unclear. We show in this study that an over-activated ATR/CHK1 pathway regulates the strong S and G2 checkpoints in Fhit−/− cells, thus contributing to the radioresistance of Fhit−/− cells, and suggesting an association between these phenotypes and tumorigenesis. Normally activated checkpoints survey DNA damage in cells and slow down cell cycle progression, thus facilitating DNA repair. Under this condition, cells with critical gene mutations and chromosomal abnormalities will die, keeping a biological balance in mammalian cells. However, if checkpoints are over-activated following DNA damage, the cells that carry gene mutations and chromosomal abnormalities will have more opportunities to survive, therefore, increasing the potential to develop into malignancies. The results that Fhit deficient cells are significantly resistant to UVC with higher mutation rates (Ottey et al., 2004) support this hypothesis. Without Fhit, the cells have a larger fraction of S phase cells than their wild type counterparts, suggesting an activated S checkpoint exists in Fhit−/− cells. This might be one of the reasons for the over-activated ATR/CHK1 pathway in irradiated Fhit−/− cells. The over-activated ATR/CHK1 pathway is responsible for the radioresistance of Fhit−/− cells, which results in more cell survival in the presence of gene mutation and chromosome abnormality from IR-induced DNA damage.
We thank Nancy Mott for help in the preparation of the article and Peggy Mammen for help in the laboratory work.