The Chinese hamster FANCG/XRCC9 mutant NM3 fails to express the monoubiquitinated form of the FANCD2 protein, is hypersensitive to a range of DNA damaging agents and exhibits a normal level of spontaneous sister chromatid exchange

James B. Wilson1, Mark A. Johnson1,5, Anna P. Stuckert2, Kelly L. Trueman1, Simon May1,6, Peter E. Bryant3, Raymond E. Meyn4, Alan D. D'Andrea2 and Nigel J. Jones1,7

1 Mammalian DNA Repair Laboratory, School of Biological Sciences, Donnan Laboratories, University of Liverpool, Liverpool, L69 7ZD, UK,
2 Department of Pediatric Oncology, Dana-Farber Cancer Institute and Department of Pediatrics, Children's Hospital, Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA,
3 School of Biomedical Sciences, University of St. Andrews, St Andrews, KY16 9TS, UK
4 Department Experimental Radiation Oncology, MD Anderson Cancer Centre, 1515 Holcombe Boulevard, Box 066, Houston, Texas 77030, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fanconi anemia (FA) is a human autosomal disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinking agents such as mitomycin C and diepoxybutane. Six FA genes have been cloned including a gene designated XRCC9 (for X-ray Repair Cross Complementing), isolated using a mitomycin C-hypersensitive Chinese hamster cell mutant termed UV40, and subsequently found to be identical to FANCG. A nuclear complex containing the FANCA, FANCC, FANCE, FANCF and FANCG proteins is needed for the activation of a sixth FA protein FANCD2. When monoubiquitinated, the FANCD2 protein co-localizes with the breast cancer susceptibility protein BRCA1 in DNA damage induced foci. In this study, we have assigned NM3, a nitrogen mustard-hypersensitive Chinese hamster mutant to the same genetic complementation group as UV40. NM3, like human FA cell lines (but unlike UV40) exhibits a normal spontaneous level of sister chromatid exchange. We show that both NM3 and UV40 are also hypersensitive to other DNA crosslinking agents (including diepoxybutane and chlorambucil) and to non-crosslinking DNA damaging agents (including bleomycin, streptonigrin and EMS), and that all these sensitivities are all corrected upon transfection of the human FANCG/XRCC9 cDNA. Using immunoblotting, NM3 and UV40 were found not to express the active monoubiquitinated isoform of the FANCD2 protein, although expression of the FANCD-L isoform was restored in the FANCG cDNA transformants, correlating with the correction of mutagen-sensitivity. These data indicate that cellular resistance to these DNA damaging agents requires FANCG and that the FA gene pathway, via its activation of FANCD2 and that protein's subsequent interaction with BRCA1, is involved in maintaining genomic stability in response not only to DNA interstrand crosslinks but also a range of other DNA damages including DNA strand breaks. NM3 and other `FA-like' Chinese hamster mutants should provide an important resource for the study of these processes in mammalian cells.

Abbreviations: CH, Chinese hamster; CHO, Chinese hamster ovary; DEB, diepoxybutane; FA, Fanconi anemia; HAT, hypoxanthene/azaserve/thymidine; HBSS, Hank's balanced salt solution; MMC, mitomycin C; PEG, polyethylene glycol; SCES, sister chromatid exchanges; SDS, sodium dodecyl sulphate; TOR, thioguanine/ouabain resistant.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fanconi anemia (FA) is an autosomal recessive genetic disease clinically characterized by progressive aplastic anemia, multiple congenital abnormalities and a predisposition to malignancy including acute myeloid leukemia and squamous carcinomas of the head and neck (13). FA displays cellular and chromosomal hypersensitivity to chemicals that induce interstrand DNA crosslinks, such as mitomycin C (MMC) and diepoxybutane (DEB). FA is regarded as a chromosome instability syndrome and the production of chromosomal damage by DEB is used as a diagnostic test for FA (4). Hypersensitivity to ionizing radiation and the radiomimetic compound bleomycin has also been reported in FA, although this is less well established than their sensitivity to crosslinking agents and may vary between complementation groups (58). FA is genetically heterogeneous, with complementation analysis based on cell fusion having established the existence of at least seven groups representing genes termed FANCA to G (9,10). However, recently it was shown that group D represents two distinct genes FANCD1 and FANCD2 (11). Six of the FA genes (FANCA, FANCC, FANCD2, FANCE, FANCF and FANCG) have currently been cloned, either by positional cloning or by cDNA isolation following functional complementation of human FA cell lines (1117). A human cDNA designated XRCC9 (X-ray Repair Cross Complementing 9) was cloned by functionally complementing a Chinese hamster ovary (CHO) mutant termed UV40 (18,19) and it was subsequently demonstrated that the XRCC9 gene is identical to the FANCG gene (15).

The FANCA, C, G and F proteins assemble in a multiprotein nuclear complex in normal cells (2022) and it was recently demonstrated that the FANCE protein interacts with FANCC, FANCA and FANCG proteins indicating that it is part of the FA protein complex (23). The formation of the nuclear complex is disrupted in cells from all FA complementation groups except the FA-D (both D1 and D2 cells) complementation group (24) indicating that FANCD1 and FANCD2 proteins function downstream or independently of the FA protein complex (11). Garcia-Higuera et al. (25) have shown that the assembled FA nuclear complex is required for the activation of the FANCD2 protein to a monoubiquitinated isoform. In normal cells, this activated FANCD2 protein co-localizes with the breast cancer susceptibility protein BRCA1 in DNA damage-induced foci and in the synaptonemal complexes of meiotic chromosomes. These observations demonstrate that the FA proteins cooperate in a cellular pathway that is activated in response to DNA damage and is involved in genome stabilization. Unlike FANCA, C, E, F and G, that have no strong homologs in non-vertebrate species, the FANCD2 protein is highly conserved in Arapidopsis, Drosophila and C.elegans (11).

Chinese hamster cell mutants hypersensitive to DNA damaging agents have proved important models for the genetic and biochemical analysis of human DNA repair and recombination processes (26,27). The availability of Chinese hamster (CH) mutants hypersensitive to ultraviolet or ionizing radiation resulted in the molecular cloning of a number of human repair genes designated ERCC (Excision Repair Cross Complementing) or XRCC (X-ray Repair Cross Complementing) involved in maintaining genome stability. These include ERCC2/XPD, ERCC3/XPB, ERCC4/XPF and ERCC5/XPG required for nucleotide excision repair and involved in the cancer-prone syndrome xeroderma pigmentosum (26). The genes, XRCC2 and XRCC3, RAD51-family members involved in homologous recombination were cloned using CH mutants termed irs1 and irs1SF (28,29). The UV40 mutant, used to clone the XRCC9/FANCG gene, is around 2-fold sensitive to ultraviolet and ionizing radiations and exhibits extreme sensitivity to mitomycin C (18). A number of other CH mutants also display marked sensitivity to MMC including irs1 (XRCC2-mutated), irs1SF (XRCC3), UV20 (ERCC1), UV41 (ERCC4), irs3 (putative XRCC10), VC8 (putative XRCC11) VH4 and CL-V5B and represent a large number of distinct complementation groups (3134). Due to their phenotypic similarities to FA cells, it has been proposed that the latter four mutants (each representing a separate complementation group) may, like UV40, be defective in the FANC gene pathway (31,32,35,36). However, despite being used to clone the human FANCG gene, doubts over the suitability of UV40 as a mammalian model for FA persist because of phenotypic differences between it and human FA cell lines, particularly the 3- to 4-fold elevated level of spontaneous sister chromatid exchanges observed in the CHO mutant (18,19).

Here we report on the complementation analysis of a mutant termed NM3. NM3 was isolated from the CHO cell line AA8 by Meyn et al. (37) on the basis of its 7-fold hypersensitivity to the crosslinking agent nitrogen mustard. It exhibits sensitivity to MMC and, like some other MMC-hypersensitive mutants such as irs1, VC8 and UV40, is approximately 2- to 3-fold hypersensitive to UV and gamma-rays (37). Our studies establish that NM3 belongs to the same complementation group as UV40. Transfection of the human FANCG/XRCC9 cDNA into NM3 functionally complements its DNA damaging agent hypersensitivities (including MMC, DEB, bleomycin and EMS) confirming the complementation group assignment. Significantly, the spontaneous levels of SCEs in NM3 are, as observed in human FA cell lines, normal. Furthermore, we demonstrate that NM3 (and UV40), like human FANCG cell lines, fail to express the monoubiquitinated form of the FANCD2 protein.


    Materials and methods
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 Introduction
 Materials and methods
 Results
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 References
 
Cells and culture conditions
The parental wildtype cell line AA8 and the AA8-derived mutants NM3 and UV40 have been described previously (18,37,38). The cell line V79 was used as an additional wildtype control for some experiments (39). Cells were routinely maintained in Dulbecco's modified Eagle's media (D-MEM, Gibco BRL, Paisley, UK) with Glutamax, supplemented with 10% fetal calf serum (Harlan Sera Laboratories) and either 100 units/ml penicillin and 100 mg/ml streptomycin sulphate or 50 µg/ml gentamicin (Gibco BRL, Paisley, UK) Cells were grown at 37°C under 5% CO2. Trypsinization was performed with 0.12% trypsin and 0.008% EDTA (T/E).

Survival curves and drug treatments
These were performed as previously described (40,41), with the exception of the EMS (ethyl methane sulphonate) and MMS (methyl methane sulphonate) treatments. Exponentially growing cells were plated into 10 cm petri dishes, and following 2 h for attachment chemical added. Five control dishes, without chemical, were prepared at 200 or 300 cells/dish; those with chemical were prepared in triplicate with increasing cell numbers used at higher doses. Preparation and storage of EMS and MMS was as previously described (42) and exposure was for 1 h in HBSS, following cell attachment. All chemicals used for survival determinations were obtained from Sigma-Aldrich, Poole, UK. After 7–10 days to allow for colony formation, dishes were fixed, stained and colony numbers determined. Each survival curve represents the mean of a minimum of three experiments and the data were fitted on a semi-log plot and sensitivity quantified by determining the D37 (dose required to reduce survival to 37% of control) for each cell line.

Cell lines and selection of TOR clones for cell fusion and hybrid formation
Hybrids were formed between pairs of cell lines using the thioguanine/ouabain resistant (TOR) hybridization and hypoxanthine/azaserine/thymidine (HAT)/ouabain selection system previously described (34,43,44). The TOR clone of NM3 was isolated by selecting spontaneous mutants as previously described (34). Cell lines irs1TOR (43) and MC5TOR were isolated previously (34), whilst TOR versions of UV40, UV20, UV41, irs1SF and VC8 were supplied by Dr David Busch, Prof. John Thacker and Dr Margaret Zdzienicka.

Cell fusion was performed using polyethylene glycol (PEG) 1000 at 50% (w/v) in Hank's balanced salt solution (HBSS) in 0.15 M HEPES, at pH 7.6. The procedure for cell fusion and isolation of hybrids is described in detail elsewhere (40) along with controls to ensure that mutations to ouabain resistance (in unmarked cell lines) or reverse mutation to HAT resistance/ 6-thioguanine sensitivity (in TOR lines) occur at a very low frequency when compared with hybrid formation, and to ensure cell line status (34). Pooled population of hybrids were maintained in HAT/ouabain medium and the survival responses of the hybrid populations were then determined as described above.

Transfection of NM3 and UV40 with XRCC9/FANCG gene
The human XRCC9/FANCG cDNA (19) was kindly provided by Dr K.J.Patel (MRC LMB, Cambridge) cloned into the pcDNA3 vector (Invitrogen, The Netherlands), which confers ampicillin resistance in E. coli and neomycin/G418 resistance in mammalian cells. For transfection a 75 cm2 flask was seeded with ~2 x 106 cells in normal growth medium and incubated overnight. Prior to transfection, 100 µg of vector DNA and 100 µl of lipofectAMINE (Gibco BRL) were added to separate 3 ml aliquots of Optimem-1 medium (Gibco BRL), mixed well and left at room temperature for 30 min. The two aliquots were then combined, mixed well and left at room temperature for a further 15 min to allow DNA/lipofectAMINE complex formation. A further 6 ml of fresh optimem medium was added, mixed and 6 ml of this added to the cells in previously drained 75 cm2 flasks. The cells were incubated in the DNA/lipofectAMINE/optimem medium at 37°C for 5 h, following which it was removed and the cells washed with HBSS. Fresh normal growth medium was added and the flasks incubated overnight. Following the transfection procedure cells were harvested and spread as follows; 200/dish as viability controls, 10 000/dish into G418 (1.5 mg/ml) to measure transfection frequency and into G418 and 50 nM MMC to enable co-selection of neo marker and FANCG/XRCC9 (a dose of 50 nM MMC gives ~70% survival for AA8). Following selection, a number of FANCG transfectants were picked and grown to high density to enable their survival responses relative to AA8, NM3 and UV40 to be determined. NM3-FANCG and UV40-FANCG transfectants were cultured in medium containing 900 mg/ml G418 in order to maintain selective pressure.

Detection of FANCD2 isoforms by immunoblotting
The generation of an affinity-purified rabbit polyclonal antiserum against FANCD2 has been described previously (25). Cells were grown to near-confluence in 175 cm2 peel-back flasks (Helena Bioscience, Sunderland, UK) and then washed extensively with HBSS. Cells were lysed with protein extraction buffer (50 mM Tris–HCl, pH 6.8, 1% sodium dodecyl sulphate (SDS), 1% EDTA, 0.25% glycerol, 0.25% ß-mercaptoethanol) and the lysate boiled for 10 min. Following centrifugation for 10 min the supernatant was taken and subjected to polyacrylamide SDS gel electrophoresis. Proteins were transferred to nitrocellulose using a submerged transfer apparatus (Wolf Labs). filled with 25 mM Tris base, 192 mM glycine and 20% methanol. After blocking with 5% non-fat dried milk in TBS-T (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Tween 20) the membrane was incubated with the anti-FANCD2 polyclonal antibody diluted in TBS-T (1:1000 dilution). After washing extensively the membrane was incubated with anti-rabbit horseradish peroxidase-linked secondary antibody and chemiluminescence used for detection (Amersham, Little Chalfont, UK). Protein size was estimated using Full Range Rainbow (Amersham) molecular weight markers (10–250 kD).

Determination of spontaneous levels of sister chromatid exchanges (SCEs)
These were determined using the fluorescence plus Giemsa (FPG) technique originally described by Perry and Wolff (45). Cells from exponentially growing cultures, were plated in 0.5 ml directly onto microscope slides at 0.5–2.0 x104 per slide and allowed to attach. Five slides per cell line were set up in square petri dishes and following the addition of a further 27.5 ml growth media incubated for ~40 h. BrdU (Sigma) was added at a final concentration of 10 µg/ml and cells incubated a further 25–30 h to allow two cycles in the presence of BrdU. Colcemid (0.1 µg/ml) was then added for 2–3 h to accumulate metaphase cells. Slides were rinsed in HBSS and placed in hypotonic solution (0.56% w/v KCl) for 5–8 min. The slides were then processed through three changes of fixative (methanol:acetic acid, 3:1) before being allowed to air dry. Slides were then immersed in Hoechst 33258 (20 µg/ml) for 10 min and then covered with 2X SSC in a square petri dish and placed under a UV lamp (366 nm) at a distance of 15 cm for 4 h. Slides were washed thoroughly in distilled H2O and allowed to dry before staining with 4% Giemsa. Cells were viewed under oil immersion and 25 metaphases were scored for presence of SCEs.


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 Materials and methods
 Results
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 References
 
Complementation analysis of NM3
NM3 was shown to exhibit an increased sensitivity to mitomycin C (~4-fold), therefore this agent was chosen for the complementation group assignment of the mutant as it has been used in previous analyses (32,34). Given that NM3 is cross-sensitive to ionizing and ultraviolet radiations, extremely MMC hypersensitive hamster mutants that are also cross-sensitive to one or both of these agents were chosen for the initial analysis. The responses of hybrids formed between NM3 and irs1 (XRCC2-mutated), irs1SF (XRCC3), UV20 (ERCC1), UV41 (ERCC4/XPF), VC8 (putative XRCC11 gene), UV40 (XRCC9/FANCG) and MC5 (an MMC-sensitive mutant that represents a new complementation group; refs 38, 46 and unpublished) are given in Table IGo in the form of D37 values. Clear complementation was observed in hybrids formed between NM3 and irs1, irs1SF, UV20, UV41, VC8 and MC5. The D37 values for these hybrids are similar to the parental cell lines of the mutants, AA8 (parent of NM3, irs1SF, UV20, UV41, MC5) and V79 (irs1, VC8) and clearly higher than that of the mutant lines themselves (NM3TOR x MC5 shown in Figure 1Go). Although full survival curves were not constructed for the TOR lines of irs1, irs1SF, UV20 and UV41 it was confirmed that survival at 20 nM MMC was <1% for these cell lines (data not shown). The response of the hybrid irs1SFTOR x NM3 was somewhat intermediate between AA8 and NM3, although Thacker and Wilkinson (47) did also show that irs1SF displayed a similar semi-dominant phenotype with respect to ionizing radiation in complementing hybrids. In contrast were the survival responses of the hybrids UV40TOR x NM3 and NM3TOR x UV40 (Table IGo and Figure 1Go). The D37 values for these two reciprocal hybrids were identical or similar to that of NM3 clearly indicating that NM3 belongs to the same complementation group as UV40. As NM3 and UV40 do show differing levels of sensitivity to MMC (4.3-fold and 17-fold respectively with respect to D37 of AA8) this complementation analysis was repeated using another crosslinking agent diepoxybutane (DEB), to which the two mutants show a much more similar response (3-fold and 4.2-fold for NM3 and UV40 respectively). Again the responses and D37 values of the two reciprocal hybrids were similar to that of NM3 confirming the assignment of NM3 to the same complementation group as UV40 (Table IGo).


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Table I. D37 values for hybrid populations and cell lines used in complementation analysis
 


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Fig. 1. Mitomycin C survival responses of AA8, NM3, UV40 and hybrids NM3TORxUV40, UV40TOR x NM3 and NM3TOR x MC5. Survival curves for AA8, NM3 and UV40 represents the mean of at least three independent experiments. Survival responses of the hybrids represent a single determination.

 
Sensitivity of NM3 and UV40 to DNA damaging agents
Table IIGo gives the D37 values of AA8, NM3 and UV40 for several DNA damaging agents. NM3 shows a 4.3-fold increase in sensitivity to MMC compared with the parental line AA8 (Figure 1Go). This is in contrast to the much greater sensitivity of the UV40 mutant (17-fold) shown here and previously (18). A similar differential between NM3 and UV40 was observed for another DNA crosslinking agent melphalan (3-fold and 9.7-fold respectively), although in broad terms more similar responses to the two other crosslinking agents DEB and chlorambucil were exhibited, with UV40 again being the most hypersensitive (survival curves for DEB shown in Figure 2AGo). In contrast was the result for the radiomimetic compound bleomycin (Figure 2BGo), to which NM3 was much more hypersensitive (7.5-fold) than UV40 (3.6-fold). Both mutants were about 2- to 3-fold sensitive to the phenyl pyridylquinoline streptonigrin and the topoisomerase I inhibitor camptothecin, whilst only a slight sensitivity (~1.5-fold) was observed to the topoisomerase II inhibitor, etoposide. Sensitivity to monofunctional alkylating agents (~3-fold for EMS and 5- to 7-fold for MMS) was observed in both mutants.


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Table II. Sensitivities of NM3 and UV40 to genotoxic agents and their correction following transfection of the human FANCG cDNA
 


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Fig. 2. Diepoxybutane (DEB) and bleomycin responses of AA8, NM3, UV40 and the transformed cell lines NM3-FANCG and UV40-FANCG. Each survival curve represents the mean of four to eight independent experiments. The standard error of the mean (SEM) was <40% of the mean for all data points shown, other than for AA8 for which the SEM was <30% of the mean.

 
Sensitivity of FANCG/XRCC9 transformants of NM3 and UV40 to DNA damaging agents
To confirm the complementation analysis NM3 (and UV40) were transfected with the human FANCG/XRCC9 cDNA. FANCG/XRCC9 pcDNA3 transformants in the G418 and MMC co-selection were obtained at a frequency of 0.2% for NM3 and 0.12% for UV40 (after correction for viability). Several corrected clones of each cell line were picked, grown to bulk and frozen. The responses of a FANCG-transformant of NM3 and UV40 are presented in Table IIGo and expressed as fold increase in sensitivity compared with AA8, alongside the same data for NM3 and UV40. Correction of the various sensitivities of NM3 and UV40 was incomplete for most of the genotoxic agents tested although the sensitivity of the transformants was slight compared with AA8 and much less than observed in the respective mutant (2.1-fold or less). For example, for MMC and DEB (Figure 2AGo) the NM3-FANCG and UV40-FANCG cell lines were 1.2–1.4 more sensitive than AA8 (in addition, similar responses were observed for two further FANCG-transformants of NM3 and UV40 with both these agents; data not shown). The sensitivity of the NM3-FANCG and UV40-FANCG transformants were similar for all agents regardless of the differing sensitivities observed in the two mutants. For example, UV40 is much more sensitive to MMC than NM3 (17-fold and 4.3-fold respectively), whilst NM3 is more bleomycin sensitive than UV40 (7.5-fold and 3.6-fold), yet the transformants exhibit similar responses to each other (Figure 2BGo and Table IIGo).

Expression of FANCD2 in NM3, UV40 and FANCG-transformants
The antibody to the human FANCD2 protein detects two isoforms in the immortal wildtype Chinese hamster cell lines AA8 and V79 (Figure 3Go). These hamster FANCD2 proteins correspond to the short form (FANCD2-S) and the long form (FANCD2-L) detected in normal human cell lines (25). The two hamster proteins run at the same position as the two human isoforms (data not shown) as detected in a human FA cell line PD-20F (FA-D2) that is functionally complemented with the FANCD2 gene (kindly provided by Barbara Cox and Markus Grompe; 11). Under the conditions described here the size of the two hamster proteins are indistinguishable from the two human isoforms and are estimated to be 155 kD (FANCD2-S) and 162 kD (FANCD2-L). In the mutants NM3 and UV40 the larger 162 kD FANCD2-L band was absent and both mutants only expressed the short form of FANCD2 (Figure 3Go, lanes 1 and 6). However, the transformants NM3-FANCG and UV40-FANCG clearly show both isoforms (Figure 3Go, lanes 3 and 5) indicating that expression of the FANCD2-L isoform was restored although not to the same level as observed in AA8. Seven independent protein extractions from NM3, UV40, NM3-FANCG, UV40-FANCG and AA8 were made and similar results obtained for each. No FANCD2-L was ever observed in NM3 and UV40 whilst it was always detected in the two transformants. In addition, it was shown that the hybrids UV40TOR x NM3 and NM3TOR x UV40 only expressed the FANCD2-S isoform (data not shown).



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Fig. 3. Expression of the two FANCD2 isoforms as detected by immunoblotting. Whole cell extracts were prepared from the cell line indicated and cellular proteins were immunoblotted with an anti-FANCD2 antiserum. Wildtype cells (AA8 and V79) express two isoforms of the FANCD2 protein, FANCD2-S (155 kDa) and FANCD2-L (162 kDa). FANCG/XRCC9 mutants NM3 and UV40 only express the FANCD2-S isoform (lanes 1 and 6). When transformed with the human FANCG cDNA, expression of the FANCD2-L isoform is restored (lanes 3 and 5).

 
Spontaneous levels of SCEs in NM3, AA8 and NM3-FANCG
Table IIIGo gives the spontaneous levels of SCEs observed in these three cell lines. No elevation of spontaneous SCEs was observed in NM3 with similar levels observed in all three cell lines.


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Table III. Frequency of spontaneous sister chromatid exchange in AA8, NM3 and NM3-FANCG
 

    Discussion
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 Abstract
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 Materials and methods
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The CHO mutant NM3 has been assigned to the same complementation group as UV40 and is therefore established as the second representative of the hamster XRCC9/FANCG complementation group. The pattern of cross-sensitivity to various DNA damaging agents of the two mutants appears to be similar, although NM3 differs from UV40 in displaying normal levels of spontaneous SCEs. In addition to their sensitivity to the interstrand crosslinking agent MMC, both mutants were previously shown to be ~2 to 3-fold sensitive to ionizing radiation and UV (18,37). Here we show that both NM3 and UV40 are hypersensitive to additional agents including both DNA crosslinking and non-crosslinking chemicals. There is variation in the level of sensitivity between the crosslinking agents (cf. DEB and chlorambucil), with UV40 being much more sensitive to MMC and melphalan than NM3 (Table IIGo). The variation in sensitivity to the different crosslinking agents may possibly be related to the fact that these agents form adducts at different sites in the DNA (48,49). In addition, the nature of the underlying FANCG mutations in the two cell lines may play a role in the differential sensitivities of the two mutants to the various DNA damaging agents. In general terms UV40 showed the greater sensitivity, although NM3, was more sensitive to the oxidative mutagens bleomycin and streptonigrin. Bleomycin is a radiomimetic compound that induces DNA single- and double-strand breaks, whilst streptonigrin is a phenyl pyridylquinoline that undergoes bioreactivation to generate hydroxyl radicals (50,51). Given that both mutants are also 2-fold sensitive to ionizing radiation it would appear they are defective in their response to certain types of DNA strand breaks in addition to DNA inter-strand crosslinks. The sensitivity of NM3 and UV40 to monofunctional alkylating agents (EMS and MMS) and camptothecin would suggest these might include single-strand breaks or replication associated DNA double-strand breaks (52). NM3 and UV40 were only slightly hypersensitive to the non-intercalating topoisomerase inhibitor etoposide (which induces protein associated DNA double strand breaks) and therefore unlike CH mutants representing pathways of double-strand break repair by NHEJ (non-homologous end joining; e.g. XR1, XRCC4-mutated) or homologous recombination (e.g. irs1, XRCC2-mutated) (35,40,53).

That the various sensitivities of NM3 and UV40 reported in this paper are a result of a defect in FANCG is confirmed by the observed correction of these sensitivities when the mutants are transfected with the human FANCG cDNA (Table IIGo). Whilst completely full complementation was not observed in the transformants or previously for MMC and UV40 (19), there could be a number of reasons for this. For example, differences in the hamster versus human proteins or in the level of expression of the protein may be plausible explanations. Certainly, there appears to be a lower level of expression of the FANCD2-L isoform in the FANCG-transfectants of NM3 and UV40 compared with AA8. Incomplete correction of mutagen-sensitivity of a hamster cell line by a complementing human gene is certainly not an unusual observation, for example human XRCC2 cDNA and irs1 (28).

It would appear that the 3- to 4-fold elevated levels of SCEs previously observed in UV40 (18,19) are not a result of the FANCG mutation in this cell line. We found normal spontaneous levels of SCEs in both NM3 and the FANCG-complemented NM3 cell line. Transfection of the FANCG/XRCC9 gene into UV40 was previously shown to fail to correct this endpoint (19), whilst other features including expression of FANCD2-L are corrected (Table IVGo). This would seem to indicate that a mutation in a gene other than FANCG is responsible for the elevated levels of SCEs in UV40. In this context NM3 may be regarded as more `FA-like' than UV40.


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Table IV. Cellular phenotype and expression of FANCD2-L isoform in cell lines
 
Hypersensitivity to non-crosslinking agents such as ionizing radiation, bleomycin and MMS has not been generally recognized as a feature of human FA cell lines, although there are reports of sensitivity to ionizing radiation and monofunctional alkylating agents (57,54,55). Carreau et al. (8) recently reported increased sensitivity to the X-ray mimetic compound bleomycin in cells from complementation groups D to G and in a further cell line (previously designated as FA-H) that is now assigned to group A (10). In addition, this `FA-H' (EUFA173) cell line, like NM3 and UV40, showed increased sensitivity to the monofunctional alkylating agents EMS and MMS. Differences in the spectrum of sensitivities between human and hamster FA cell lines may reflect differences in the specific gene mutations/alleles involved, with the possibility that the hamster cell lines may have mutations not ordinarily observed in the human situation. CH mutants of the NER gene ERCC4/XPF, a highly conserved gene, often exhibit extreme hypersensitivity (~60-fold) to MMC, a feature not observed in human xeroderma pigmentosum cell lines (26). There may also be more variation than anticipated between, and possibly within, the various FA complementation groups (cf. responses of the cell line EUFA173 with other human FA-A cell lines). Certainly, only small numbers of human lines of defined complementation groups have been tested for their sensitivity to agents other than MMC and DEB. This issue of allelic variation may be resolved by examining the sensitivities of the various mouse FA gene knockout cell lines that are becoming available (56). The responses of the two hamster FANCG mutants reported here and previously, together with the data of Carreau et al. (8) do indicate that, in addition to the characteristic sensitivity to crosslinking agents, mammalian FA cells may also be sensitive to a much broader spectrum of DNA damaging agents. The idea that the FA gene pathway is involved in responding to DNA damage other than DNA crosslinks is supported by the recent observations made regarding the FANCD2 protein (25). Here it was shown that MMC, ionizing radiation and UV all activated a time-dependent and dose-dependent conversion of FANCD2-S to FANCD2-L as well as an increase in FANCD2 foci. It has also been reported that human FA cell lines are deficient in the NHEJ of double-strand breaks (57).

Transfection of the human FANCG cDNA restored the ability of NM3 and UV40 to convert the short form of FANCD2 protein to its monoubiquitinated form (Figure 3Go). Like human FANCG cell lines, both NM3 and UV40 fail to form the protein complex (unpublished data) and only express the FANCD2-S isoform. Thus, functional complementation of the sensitivities of NM3 and UV40 coincides with the expression of the FANCD2-L isoform, indicating that cellular resistance to these DNA damaging agents is mediated via the FANC gene pathway including the FANCG gene. The FANCG protein is 65 kDa, 622 amino acids in length and has been shown to directly interact with the FANCA, FANCE and FANCF proteins and is required for the binding of FANCC in the complex (22,23,57,58). FANCG binds to the amino terminal FANCA NLS (nuclear location signal) sequence and FANCA and FANCG stabilize each and promote the nuclear accumulation of the FA complex (58,59). A direct interaction of FANCF with FANCG has been demonstrated together with a weaker interaction of FANCE with FANCG (22,23). Also, amino acid sequences at the carboxy terminus of FANCG are required for binding of FANCC in the complex (59). These interactions indicate a critical role for FANCG in the FA complex and its formation, and it has been shown that FA-G patients have more severe cytopenia and a higher incidence of leukemia than other FA complementation groups (60,61). Recently it was demonstrated that FANCG is a phosphoprotein and is upregulated with FANCA after TNF-alpha treatment (62).

Our results indicate that hamster FANCG mutants are hypersensitive to a broader range of DNA damaging agents than simply DNA crosslinking agents. Complementation by the human FANCG cDNA corrects these sensitivities and restores expression of the long monoubiquitinated form of the FANCD2 protein. Thus it would appear that the FA gene pathway, via its activation of FANCD2 and its subsequent interaction with BRCA1, is involved in maintaining genomic stability in response to a range of DNA damages including DNA crosslinks and strand breaks. The analysis of CH cell mutants defective in this pathway is likely to provide a useful adjunct to the human studies in dissecting these processes. Finally, screening of additional hamster cell lines for expression of the FANCD2 isoforms by anti-FANCD2 immunoblotting is likely to identify further mutants defective in this critical pathway.


    Notes
 
5 Current address: MRC Laboratory of Molecular Biology, Cambridge, CB2 2QH, UK Back

6 Current address: NWG Biotech UK Ltd., Milllcourt, Featherstone Road, Wolverton Mill South, Milton Keynes MK12 5RD, UK Back

7 To whom correspondence should be addressed Email: njjones{at}liv.ac.uk Back


    Acknowledgments
 
This work was supported by project grants from the North West Cancer Research Fund (NWCRF-CR527 and NWCRF-CR386) to NJJ, a BBSRC research studentship (MAJ) and a University of Liverpool RDF grant (Ref. 4102-Dempster Bequest). We would like to thank Ellen Rushton, Greg Fitzgibbon and Helen Budworth for performing a number of the survival assays, K.J.Patel and Larry Thompson for the FANCG/XRCC9 cDNA vector and those individuals who supplied cell lines, particularly David Busch who provided UV40 and UV40TOR.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 29, 2001; revised August 3, 2001; accepted August 21, 2001.