A Role for the Fanconi Anemia C Protein in Maintaining the DNA Damage-induced G2 Checkpoint*

Fanconi anemia (FA) is a complex, heterogeneous genetic disorder composed of at least 11 complementation groups. The FA proteins have recently been found to functionally interact with the cell cycle regulatory proteins ATM and BRCA1; however, the function of the FA proteins in cell cycle control remains incompletely understood. Here we show that the Fanconi anemia complementation group C protein (Fancc) is necessary for proper function of the DNA damage-induced G2/M checkpoint in vitro and in vivo. Despite apparently normal induction of the G2/M checkpoint after ionizing radiation, murine and human cells lacking functional FANCC did not maintain the G2 checkpoint as compared with wild-type cells. The increased rate of mitotic entry seen in Fancc–/–mouse embryo fibroblasts correlated with decreased inhibitory phosphorylation of cdc2 kinase on tyrosine 15. An increased inability to maintain the DNA damage-induced G2 checkpoint was observed in Fancc –/–; Trp53 –/–cells compared with Fancc –/–cells, indicating that Fancc and p53 cooperated to maintain the G2 checkpoint. In contrast, genetic disruption of both Fancc and Atm did not cooperate in the G2 checkpoint. These data indicate that Fancc and p53 in separate pathways converge to regulate the G2 checkpoint. Finally, fibroblasts lacking FANCD2 were found to have a G2 checkpoint phenotype similar to FANCC-deficient cells, indicating that FANCD2, which is activated by the FA complex, was also required to maintain the G2 checkpoint. Because a proper checkpoint function is critical for the maintenance of genomic stability and is intricately related to the function and integrity of the DNA repair process, these data have implications in understanding both the function of FA proteins and the mechanism of genomic instability in FA.

Fanconi anemia (FA) 1 is an autosomal recessive disorder characterized by a range of congenital abnormalities, progres-sive bone marrow failure, and a propensity to develop myeloid leukemia as well as epithelial and other cancers (1,2). FA cells show a characteristic pattern of hypersensitivity to bifunctional alkylating agents such as mitomycin C or diepoxybutane (3). This hypersensitivity was utilized in cell fusion studies to identify 11 complementation groups (FAA-L). Eight of these gene products (for review, see Refs. 4 and 5) have been cloned. Six FA proteins (FANCA, FANCC, FANCG, FANCE, FANCF, and FANCL) are assembled within a nuclear complex (6 -10). The FA complex was shown to be necessary to conjugate a single ubiquitin residue to FANCD2, a process that is required for functional activity of the FA complex (11,12).
The propensity for chromosomal breaks in FA cells and the activation of the FA complex by DNA-damaging agents implicates these proteins in the DNA damage response. Despite recent advances, the precise role of the FA proteins in maintaining genomic integrity is not well understood. An emerging body of evidence implicates the FA proteins in the response of cells to double strand DNA (dsDNA) breaks. The FA complex is activated by ionizing radiation (IR) (12), which induces doublestrand breaks. FANCD2 is recruited to foci in a BRCA1-dependent manner in response to IR (12), and these foci may be sites of DNA breaks and their repair. Additionally, recent studies indicate that the FA proteins colocalize with Rad51 foci (13) and may be involved in the repair of double-strand breaks by homologous recombination (14 -16). Homologous recombination repair is active during S and G 2 phases of the cell cycle (17)(18)(19) and is closely linked to S-and G 2 -phase cell cycle checkpoints (20 -22). DNA repair and checkpoints are highly integrated processes that are important for maintaining genomic integrity (for review, see Ref. 23).
A number of studies demonstrate that FA proteins functionally and physically interact with effectors of S and G 2 cell cycle checkpoints (12,24,25), and studies have implicated a defective S-phase checkpoint response in FA cells (25)(26)(27)(28). This includes the previously mentioned BRCA1, which is required for proper G 2 checkpoint function (29 -31). Also the ATM kinase, which is a master regulator of the S and G 2 checkpoint, directly phosphorylates FANCD2 (25). Furthermore, one recent study implicates the ATR kinase in the activation of FANCD2 in response to cross-linking agents (28).
Although these studies indicate that FA proteins interact functionally with biochemical pathways that regulate checkpoint control, the role of the FA proteins in checkpoint function is incompletely studied. To test the hypothesis that the FA proteins function in G 2 checkpoint regulation, we measured the IR-induced G 2 checkpoint in primary murine and human cells lacking functional FANCC using in vitro and in vivo analyses.
We report that the G 2 checkpoint was induced normally but not maintained in cells lacking functional FANCC (Fancc) after treatment with IR. Immortalized cells lacking FANCD2 exhibited a similar phenotype. The inability of cells to maintain the G 2 checkpoint correlated with an inability to maintain tyrosine 15 phosphorylation on cdc2 despite apparently normal induction of the Atm/Chk2 pathway. Utilizing genetic intercrosses, we found that the G 2 checkpoint defect in AtmϪ/Ϫ; FanccϪ/Ϫ cells was similar to AtmϪ/Ϫ cells, whereas FanccϪ/Ϫ; Trp53Ϫ/Ϫ cells had a more severe inability to maintain the G 2 checkpoint compared with cells that were mutant at Fancc or Trp53 only. These data are consistent with Fancc functioning via a p53-independent mechanism to maintain the G 2 checkpoint. These data have implications in understanding both the function of FA proteins and the mechanism of genomic instability in FA.

EXPERIMENTAL PROCEDURES
Cell Culture-Primary murine embryo fibroblasts (MEFs) were obtained from day 12-14 mouse embryos derived from breeding of Fanccϩ/Ϫ mice. Embryos were obtained, and a small amount of tissue was saved for PCR analysis. Fibroblasts were derived by mincing embryos and culturing on tissue culture dishes in growth media consisting of Dulbecco's modified Eagle's/F-12 media containing 10% fetal calf serum supplemented with 2 mM L-glutamine, 100 units/ml penicillin and streptomycin (Invitrogen). MEFs were maintained based on a 3T3 protocol (32). All experiments using MEFs were performed with cells that were less than passage 6. Primary human skin fibroblasts were isolated from two FA complementation group C patients and cultured in Dulbecco's modified Eagle's containing 10% fetal calf serum and supplemented with 2 mM L-glutamine, 100 units/ml penicillin and streptomycin. These cells were transduced with a retrovirus expressing FANCC (33) and selected in G418. Both control and FANCD2-corrected immortalized fibroblasts from a FANCD2 patient (PD20 and PD20ϩFANCD2) were gifts of the Fanconi anemia foundation and were cultured the same as primary skin fibroblasts.
Mouse Models-Mice harboring a disruption of the Fancc gene were generously provided by Dr. Manuel Buchwald (34). These mice were backcrossed for 10 generations into the C57BL/6J strain. Mice were genotyped using PCR to detect wild-type and mutant Fancc alleles as described (35). Mice with the disrupted Atm allele have been previously described (36) and were obtained from The Jackson Laboratory (Bar Harbor, ME) in the 129 genetic background. Genotyping for Atm was carried using PCR primers as suggested by the vendor. These mice were crossed with Fancc mutant mice in the C57BL/6J strain to yield mice with a mixed genetic background. F2 embryos were generated by crossing Atm and Fancc double heterozygous animals, and MEFs were isolated from these embryos. Experiments comparing the Fancc and Atm genotypes to one another were carried out using MEF lines isolated from a common pregnant female. Mice harboring a disruption of the Trp53 gene have been previously described (37) and were obtained in the C57BL/6J strain from The Jackson Laboratory. Trp53 genotype was determined using a PCR method and primer sequences as provided by The Jackson Laboratory. Fancc and Trp53 compound heterozygous mice were generated by mating Fanccϩ/Ϫ mice with Trp53ϩ/Ϫ mice. The F2 generation was produced by crossing the compound heterozygotes derived from the F1 mice.
Treatment of Cells with IR and Mitotic Index Determination-For analyzing G 2 progression of a defined S-phase population, cells were pulse-labeled with 10 M bromodeoxyuridine (BrdUrd). Cells were then treated with ionizing radiation using a Gammacell-40 exactor (Nordion) containing a 137 Cs source and cultured for the indicated time period followed by analysis of BrdUrd incorporation. BrdUrd was detected using anti-BrdUrd FITC antibody (Pharmingen) according to the manufacturer's specifications. Briefly, cells were fixed in 70% ethanol, denatured (2 M HCl), and neutralized (0.1 M sodium borate). Cells were stained with anti-BrdUrd FITC antibody, then resuspended in 50 g/ml propidium iodide (Sigma) and analyzed on a FACScan flow cytometer (BD Biosciences). For mitotic index determination, cells were cultured on tissue culture slides (Nunc), treated with IR, and incubated for the indicated time. Slides were fixed in 100% methanol containing 2 g/ml 4,6-diamidino-2-phenyldole (Sigma) and condensed, and mitotic nuclei were visualized and scored by fluorescent microscopy. For analysis of phosphorylated histone H3, MEFs of the appropriate genotypes were treated with 10 Gy of IR, cultured for the indicated time period, then trypsinized and fixed in 70% ethanol. After fixation cells were permeabilized (0.25% Triton X-100 on ice for 15 min), washed, and incubated for 1 h in phosphate-buffered saline containing 0.1% bovine serum albumin and 0.75 g of phosphohistone H3 antibody (Upstate Biotechnology). After washing, cells were then incubated in secondary antibody (goat anti-mouse FITC, Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min at room temperature. Cells were then incubated in 50 g/ml propidium iodide and 100 g/ml RNase A for 30 min and analyzed on a flow cytometer for FITC (FL-1) and propidium iodide (FL-2). For all cells analyzed, the G 1 peak was standardized at a fluorescence intensity of 200 in FL-2.
G 2 Checkpoint Analysis in Keratinocytes-The analysis of the G 2 checkpoint was carried out in vivo as previously described (38) by measuring the percent of mitotic cells in keratinocytes in syngeneic, 3-6-month-old age and sex-matched mice. Mice were irradiated with 5 Gy of IR and then allowed to recover for the given time period. Mice were then sacrificed, and skin was fixed in 10% formalin. Processing was performed for phosphohistone H3 antibody (Upstate Biotechnology), used at a 1:500 dilution. Approximately one mitotic event was scored in untreated keratinocytes per high power field.
S-phase Checkpoint Analysis-Analysis of the IR-induced S phase checkpoint was carried out according to previously established methods. Fibroblasts were first labeled with [ 14 C]thymidine (0.5 nCi/ml) for 36 h and washed, and cells were then cultured overnight. Cells were irradiated (20 Gy) and pulse-labeled for 15 min with [ 3 H]thymidine at varying time periods after IR. Labeling was terminated (10% 2.3 M citric acid), cells were fixed (10% trichloroacetic acid), DNA was solubilized (0.2 M NaOH), and the solution was counted on a scintillation counter for both 14

Murine (Fancc) and Human (FANCC) Fibroblasts
Have an Impaired IR-induced G 2 Checkpoint-To test the hypothesis that the murine homologue (Fancc) of Fanconi anemia type C (FANCC) functions in G 2 checkpoint control, the cell cycle kinetics of a defined population of primary FanccϪ/Ϫ and Fanccϩ/ϩ MEFs were examined after treatment with ionizing radiation. We monitored the progression of a BrdUrd-labeled (S phase) population of cells through G 2 and M phases of the cell cycle after IR treatment, similar to the methodology used previously (39,40). Cells were cultured for the indicated times and analyzed for simultaneous detection of BrdUrd and DNA content using flow cytometry (Fig. 1a). The pulsed cells were arrested with primarily 4n DNA content by 8 h after IR treatment in both genotypes. Although Fanccϩ/ϩ cells remained arrested in G 2 /M, the FanccϪ/Ϫ cells began to exit G 2 /M, and a significant proportion of cells (30%) escaped from 4n arrest 12-16 h post-IR. The data are summarized in a graph depicting the results from three independently isolated MEF lines (Fig. 1b).
To determine whether the checkpoint abnormality observed in Fancc null MEFs also occurred in human cells, FANCC mutant cutaneous fibroblasts were examined for G 2 arrest after IR treatment. Fibroblasts from two FA patients with FANCC mutations were transduced with a retrovirus encoding the FANCC cDNA and neo or a retrovirus containing neo only. FANCC fibroblasts (control and FANCC-corrected) were treated with BrdUrd for 1 h, exposed to 5 Gy of IR, and cultured for up to 48 h (Fig. 1, c-d). In both patients the BrdUrd-labeled cells (both FANCC gene-corrected and FANCC mutant) were essentially arrested with 4n DNA content 8 h after IR. However, although most of the cells expressing recombinant FANCC maintained the arrest at 24 and 48 h post-IR, a much higher percentage of the patient fibroblasts expressing only neo exited G 2 and mitosis into the subsequent cell cycle. These data demonstrate that a significant portion of cells lacking functional murine Fancc and human FANCC did not maintain cell cycle arrest at 4n compared with controls expressing functional FANCC.
To further analyze the DNA damage-induced G 2 checkpoint in FanccϪ/Ϫ MEFs, we determined the mitotic index of murine Fanccϩ/ϩ and FanccϪ/Ϫ cells after treatment with IR. Compared with Fanccϩ/ϩ cells, a significantly increased percentage of FanccϪ/Ϫ cells were observed in mitosis after IR treatment (Fig. 2a). In human fibroblasts a similar reduction in mitotic index was observed after IR in the FANCC mutant cells expressing recombinant FANCC protein, whereas uncorrected FANCC mutant cells demonstrated a significantly higher rate of mitosis after IR treatment (Fig. 2b).
Since biochemical pathways exist for both the induction and maintenance of the G 2 checkpoint (41, 42), we tested whether Fancc is important for initiating or maintaining the G 2 checkpoint. Mitotic index was assessed as a function of time after treatment of Fanccϩ/ϩ and FanccϪ/Ϫ cells with IR by microscopy (Fig. 2c) and by analysis of phosphorylated histone H3 (Fig. 2, d-e), which specifically occurs during mitosis (43,44). Consistent with previous observations by others (42,45,46), we observed in Fanccϩ/ϩ cells a marked reduction in mitosis by 1 h after IR treatment that was essentially maintained for a prolonged time course (up to 12 h). Although FanccϪ/Ϫ cells demonstrated normal induction of the G 2 checkpoint, they began to exit G 2 and enter mitosis between 4 and 6 h after IR, indicating a relative inability to maintain the G 2 checkpoint as compared with Fanccϩ/ϩ cells.
To test whether the G 2 checkpoint was functional in FanccϪ/Ϫ cells in vivo, we compared the induction and maintenance of G 2 in keratinocytes from irradiated Fanccϩ/ϩ and FanccϪ/Ϫ mice. Histologic sections of skin from the irradiated mice were processed and analyzed by immunohistochemistry for phosphohistone H3 (38). Consistent with previous studies (38), a reduction in the percentage of phosphohistone H3 positive cells was observed in wild-type cells at 6 and 24 h after IR treatment (Fig. 3). In contrast, although a reduction in mitotic cells was observed in FanccϪ/Ϫ cells 6 h after IR treatment, the percentage of mitotic cells had increased to untreated levels in FanccϪ/Ϫ skin sections by 24 h after treatment. Overall these data indicate that in primary cells Fancc is dispensable for initiation but required for proper maintenance of the G 2 checkpoint.
Evaluation of cdc2 Phosphorylation in FanccϪ/Ϫ Cells after IR-A critical regulator of the G 2 /M transition is the phosphoprotein cdc2, which is highly regulated during G 2 and M phases (47). Sustained phosphorylation of tyrosine 15, which occurs as a result of DNA damage, prevents entry of cells into mitosis (47,48). We, therefore, determined whether the inability of FanccϪ/Ϫ cells to maintain the IR-induced G 2 checkpoint was associated with altered cdc2 phosphorylation (Fig. 4a, top  panel). Fanccϩ/ϩ and FanccϪ/Ϫ cells were treated with IR, and phosphorylation of cdc2 was assessed using an antiserum specific to tyrosine 15-phosphorylated cdc2 (Fig. 4a). Induction of tyrosine15 phosphorylation was observed in both Fanccϩ/ϩ and FanccϪ/Ϫ cells after IR treatment. Interestingly, although tyrosine 15-phosphorylated cdc2 was evident at all time points after IR in Fanccϩ/ϩ cells, we observed that cdc2 phosphorylation was decreased significantly by 8 h post-IR in FanccϪ/Ϫ cells. A good correlation between increased mitotic index and decreased tyrosine 15-phosphorylated cdc2 was observed in both Fanccϩ/ϩ and FanccϪ/Ϫ cells (Fig. 4b). Taken together, these data indicate that the inability of FanccϪ/Ϫ cells to maintain the IR-induced G 2 checkpoint correlated with decreased tyrosine 15-phosphorylated cdc2.
ATM is an important regulator of the G 2 checkpoint that activates a signaling cascade, resulting in accumulation of tyrosine 15-phosphorylated cdc2 after IR (42,49,50). FANCC could function downstream of ATM or in a separate pathway to maintain the G 2 checkpoint. To differentiate these possibilities, we crossed Fancc-deficient mice with Atm-deficient mice to determine whether null mutations in Fancc and Atm cooperate in loss of G 2 checkpoint control. We measured the mitotic index by phosphohistone H3 analysis in primary MEFs from the F2 progeny of this intercross (Fig. 4c). Compared with Fanccϩ/ϩ; Atmϩ/ϩ MEFs, Fanccϩ/ϩ; AtmϪ/Ϫ MEFs failed to induce a normal G 2 checkpoint, consistent with previous literature (42,45), whereas FanccϪ/Ϫ; Atmϩ/ϩ cells induced but did not maintain the G 2 check point. Cells that were FanccϪ/Ϫ; AtmϪ/Ϫ exhibited a checkpoint defect that was very similar to similar results is shown. Statistical significance was assessed by Student's t test. d, a representative analysis of cells analyzed for phosphohistone H3 staining. Cells were cultured after treatment with IR, fixed, and stained with antibody and propidium iodide (PI). The stained cells were then subjected to bivariate analysis for phosphohistone H3 in FITC (FL-1) and PI (FL-2) on a flow cytometer. e, P-H3 analysis of cells cultured for various times after IR treatment. Cells were prepared and analyzed as in d and are represented graphically as the mean percent of control phosphohistone H3 Ϯ S.E. of three independently performed experiments. Percent of control untreated cells is represented on the y axis, and time following IR treatment is shown on the x axis. Statistical analysis was carried out using a paired t test. NT, not treated; IR, treated with ionizing radiation. One important target of ATM is the checkpoint kinase CHK2 (49). To test whether Fancc is required for Atm-dependent activation of Chk2, we examined Chk2 activation in FanccϪ/Ϫ cells after IR (Fig. 4b). In both FanccϪ/Ϫ and Fanccϩ/ϩ cells, Chk2 protein was converted to the phosphorylated, slower mi-grating form after IR treatment (49), indicating normal phosphorylation of Chk2. Taken together, these data suggest that Fancc may be in an Atm-dependent pathway subsequent to Atm and Chk2 activation.
Fancc and Trp53 Cooperate in G 2 Checkpoint Control-Previous studies have demonstrated a critical function in G 2 checkpoint control for the tumor suppressor p53 since cells

FIG. 3. Analysis of mitotic index by phosphohistone H3 determination in Fancc؉/؉ and Fancc؊/؊ mice in vivo in keratinocytes.
Mice were treated with 5 Gy of IR, then allowed to recover for 6 or 24 h. Mice were then euthanized, and skin sections were prepared for analysis by immunohistochemistry for phosphohistone H3. The data shown are the mean percent of control phosphohistone H3 (ϮS.E.) of five mice for each experimental group. Statistics were performed using Students' t test.

FIG. 4. The phosphorylation of tyrosine 15 of cdc2 is not maintained in Fancc؊/؊ cells, but Atm dependent activation of Chk2 is
normal. a, cells were treated with 10 Gy of IR and cultured for the indicated time periods, and protein extracts were analyzed by Western blotting using antibodies specific to the indicated protein. Total protein levels are normalized using ␤-actin as a control. A representative of three independent experiments is shown. P-, phospho-. b, correlation of mitotic index with phosphorylated cdc2. Mitotic index values obtained from Fanccϩ/ϩ and FanccϪ/Ϫ mice were graphed in relation to phosphorylated cdc2. Tyrosine-phosphorylated cdc2 was assessed by densitometry (of blots from a) and normalized by comparison to the loading control to obtain normalized density units (right y axis). Both y parameters were graphed as a function of time after IR treatment. The curves obtained for Fanccϩ/ϩ cells are shown on the left and for FanccϪ/Ϫ cells are shown on the right. c, lack of additive cooperation between null Fancc and Atm alleles. Fancc and Atm mice were crossed to yield mice heterozygous for both alleles. Double heterozygous mice were then crossed, and MEFs from the F2 generation (ϩ/ϩ;ϩ/ϩ, FanccϪ/Ϫ;Atmϩ/ϩ, Fanccϩ/ϩ; AtmϪ/Ϫ, or Ϫ/Ϫ;Ϫ/Ϫ), were analyzed using for p-H3 by flow cytometry as done in Fig. 2d-e. Percent of control p-H3 is shown on the y axis, and time after IR treatment is shown on the x axis. The data shown are a mean (ϮS.E.) of three independently performed experiments. Statistics were performed using a paired t test. NT, not treated. lacking p53 do not maintain the G 2 checkpoint (45,46,51). Fancc may function in the G 2 checkpoint in a parallel pathway to p53, cooperating to maintain G 2 , or in a p53-dependent effector pathway. To differentiate between these two possibilities, we crossed heterozygous Fancc and Trp53 mutant mice and measured the G 2 checkpoint by mitotic index determination after IR treatment in MEFs lacking both Fancc and Trp53 (Fig. 5). The virtual absence of mitotic cells at 1 h after IR indicates a strong G 2 checkpoint induction in cells of all Trp53 and Fancc genotypes. Fanccϩ/ϩ; Trp53Ϫ/Ϫ cells began to enter mitosis at ϳ12 h with similar kinetics as FanccϪ/Ϫ;p53ϩ/ϩ cells, whereas FanccϪ/Ϫ; p53Ϫ/Ϫ cells entered mitosis at a much higher frequency at 6 -12 h after IR. Collectively, these data indicate that Fancc and p53 cooperate in maintaining the G 2 checkpoint.
Because FANCC is a component of the FA complex that is required for monoubiquitination of FANCD2, we questioned whether the role of FANCC in G 2 checkpoint control was dependent on its established function in activating FANCD2. Immortalized FA-D2 fibroblasts (PD20 cells) and cells derived from the PD20 cell line that had been corrected with FANCD2 (52) were treated with IR, and mitotic index (phosphorylated histone H3) was determined over an extended time course after IR treatment (Fig. 6). Although the G 2 checkpoint was induced in the FANCD2 mutant PD20 cells, these cells began to enter mitosis much earlier than PD20 cells corrected with FANCD2, indicating that cells lacking functional FANCD2 did not maintain the G 2 checkpoint. These findings were similar to that observed in cells lacking functional FANCC protein and indicate that the FA complex substrate FANCD2 is also required for maintaining the G 2 checkpoint. DISCUSSION Cell cycle checkpoints are a vital component of the cellular response to intrinsic and exogenous sources of DNA damage. Loss of checkpoint control results in disruption of the DNA damage response and leads to genomic instability and malignancy (for review, see Ref. 23). Here we show that cells lacking the FA protein, FANCC, initiate but do not maintain the G 2 checkpoint induced by ionizing radiation both in fibroblasts in vitro and in keratinocytes in vivo. Because checkpoints are linked to dsDNA break repair mechanisms such as homologous recombination repair (20 -22), the inability of FA cells to maintain the G 2 checkpoint may be related to disturbances in dsDNA break repair. Double-strand DNA breaks are generated by IR as well as endogenous processes such as DNA replication; therefore, abnormal G 2 checkpoint function could be a primary source of genetic instability in FA.
Fancc cooperates with p53 in the G 2 checkpoint, indicating that these proteins function in distinct pathways that converge to regulate maintenance of the G 2 checkpoint. There are several different mechanisms by which p53 functions in G 2 checkpoint control. These include p53-dependent activation of the p21 cip1/waf1 cyclin-dependent kinase inhibitor (46,51,53,54) as well as controlling subcellular localization of cyclin B and cdc2 (55,56). Our data indicate that the p53 independent role of Fancc in maintaining the G 2 checkpoint correlates with an inability of Fancc to maintain tyrosine phosphorylation on cdc2, indicating that Fancc influences cdc2 function directly or indirectly. Indirectly, altered cdc2 phosphorylation could simply be a consequence of the inability of FanccϪ/Ϫ cells to maintain G 2 arrest, as cdc2 phosphorylation is tightly correlated with mitotic entry (47,48,57). However, other studies suggest that Fancc could act directly in mediating cdc2 activity or phosphorylation. Two reports indicate that the expression of FANCC is highest in G 2 and mitosis (58,59), suggesting that FANCC may have a unique function in G 2 . Also, FANCC is reported to associate with cdc2 (58), which may imply a functional interaction.
In contrast to Fancc and p53, cooperation was not observed in cells mutant for both Fancc and Atm. ATM is an important mediator of Cdc2 activity via the CHK1/CHK2 kinases and Cdc25 phosphatases (49,50). Our genetic data indicating that Atm and Fancc do not cooperate would be consistent with Fancc being downstream of Atm in a linear pathway of G 2 checkpoint regulation. However, further characterization of the interaction between effectors in the ATM and FA pathway using biochemical means will be required to investigate this possibility. ATM was shown previously to phosphorylate FANCD2, suggesting a potential interaction between FANCC and ATM (25). Other studies indicate that ATR interacts with the FA pathway in the response to DNA cross-linking agents (28); hence, ATR could also mediate the FA protein function in the G 2 checkpoint. Because mouse cells doubly mutant for Atm and Atr exhibit cooperative defects in maintaining the G 2 checkpoint (42), one would expect that an ATR effector would also cooperate with ATM. Because Fancc did not cooperate with Atm, the data are not consistent with a role for FANCC in an ATR-dependent pathway after IR.
Emerging evidence increasingly supports a role for the FA proteins in mediating the dsDNA break response (12,16,24,60). Our data support a model in which, after FA complex activation and FANCD2 localization to foci (12), FANCD2 FIG. 6. The G 2 checkpoint is not maintained following IR in an immortalized cell line lacking functional FANCD2. The G 2 checkpoint was measured as a function of time after IR treatment by assessing the mitotic index using phosphohistone H3 analysis by flow cytometry as described under "Experimental Procedures." PD20 cells from an FA-D2 patient and PD20 cells corrected with a retrovirus expressing FANCD2 were treated with 5 Gy of IR, then fixed after culture for the indicated time period. The data are shown as percent mitotic phosphohistone H3 (y axis) as a function of hours after IR treatment (x axis). The data shown are a mean of three independent experiments. *, p Ͻ 0.01.
(alone or in combination with other FA proteins) signals the checkpoint and dsDNA break repair machinery by a yet to be determined mechanism. In fact, FancgϪ/Ϫ murine cells (alternatively, XRCC9 mutant) fail to maintain the G 2 checkpoint, similar to the defect shown here in FanccϪ/Ϫ cells, 2 indicating that other proteins in the FA complex function in this capacity, most likely by activating FANCD2. This model is supported by our observation indicating that cells lacking FANCD2 also exhibit an inability to maintain the G 2 checkpoint. Our interpretation is in contrast to that of Taniguchi et al. (25), who observed a defect in the S phase, but not in the G 2 phase checkpoint in FANCD2 mutant cells. However, the methodology employed in that study (a mitotic spindle inhibitor to trap cells that accumulate in mitosis) assessed mitosis at a single time point after IR. In contrast, evaluating the proportion of cells that escape G 2 at multiple time points using the methodology employed in the current studies allows a more detailed measurement of the cellular capacity to maintain the G 2 checkpoint.
Although FA cells are hypersensitive to cross-linking agents (3), they exhibit only a modest hypersensitivity to IR (25,61,62). DNA cross-linking agents produce bulky adducts that interfere with DNA replication in S phase, and a failure to properly resolve cross-links (or partially processed cross-links) in FA cells during S phase may account for the characteristic hypersensitivity to these agents (63). This would be consistent with homologous recombination repair-deficient cells such as the Rad51 paralogs, which have a profound hypersensitivity to cross-linking agents (64,65) and lesser hypersensitivity to IR (64). FA cells exhibited an apparently normal capacity to initiate the G 2 checkpoint, as opposed to ATM cells that do not initiate a normal G 2 checkpoint and have a profound hypersensitivity to IR (42,66,67). Furthermore, IR-induced dsDNA breaks are rapidly rejoined within 1 h in normal cells (for review, see Ref. 68), and there is no detectable defect in rejoining of breaks in FA cells. 3 We also found that FanccϪ/Ϫ cells exhibited a normal IR-induced S phase checkpoint (data not shown), consistent with previous findings in FANCC fibroblasts (25) and similar to that observed in the murine model for Fancd2Ϫ/Ϫ (69). Therefore, an S phase checkpoint defect is unlikely to contribute to the G 2 phenotype observed in the current study. However, FA proteins are activated by IR (12,24,25), and FA-deficient cells exhibit genomic instability as a result of IR (16), indicating an important role in this response. Further studies are required to determine whether Fancc-deficient cells and cells from other FA complementation groups have altered fidelity of repair in response to IR, as might be predicted based on an inability to maintain the G 2 checkpoint. Importantly, FA cells can survive low dose IR; hence, genomic instability induced by IR could persist in these cells. This could have important clinical implications since IR is utilized in conditioning regimens before bone marrow transplantation of FA patients. In conclusion, the role of FA proteins in maintaining the G 2 checkpoint has significant implications for genomic instability and in understanding the function of the FA proteins.