Download The Chinese hamster FANCG/XRCC9 mutant

Carcinogenesis vol.22 no.12 pp.1939–1946, 2001
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.Bryant 3, Raymond E.Meyn4,
Alan D.D’Andrea2 and Nigel J.Jones1,7
1Mammalian
DNA Repair Laboratory, School of Biological Sciences,
Donnan Laboratories, University of Liverpool, Liverpool, L69 7ZD, UK,
2Department of Pediatric Oncology, Dana-Farber Cancer Institute and
Department of Pediatrics, Children’s Hospital, Harvard Medical School,
44 Binney Street, Boston, MA 02115, USA, 3School of Biomedical
Sciences, University of St. Andrews, St Andrews, KY16 9TS, UK
4Department Experimental Radiation Oncology, MD Anderson Cancer
Centre, 1515 Holcombe Boulevard, Box 066, Houston, Texas 77030, USA
5Current
address: MRC Laboratory of Molecular Biology, Cambridge, CB2
2QH, UK
6Current
address: NWG Biotech UK Ltd., Milllcourt, Featherstone Road,
Wolverton Mill South, Milton Keynes MK12 5RD, UK
7To
whom correspondence should be addressed
Email: [email protected]
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 mustardhypersensitive 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
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.
© Oxford University Press
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.
Introduction
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 (1–3). 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 (5–8). 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 (11–17). 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 (20–22) 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
1939
J.B.Wilson et al.
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 (31–34). 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
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
1940
(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 ⫻ 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 nearconfluence 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% β-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
New Chinese hamster FANCG/XRCC9 mutant
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 antiFANCD2 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 ⫻104
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.
Results
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 crosssensitive 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 I 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 ⫻ MC5 shown
in Figure 1). 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 ⫻ 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 ⫻ NM3 and NM3TOR ⫻ UV40 (Table I and
Figure 1). 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
Table I. D37 values for hybrid populations and cell lines used in
complementation analysis
D37 mitomycin C (nM)
Cell line
AA8
NM3
UV40
NM3TOR
UV40TOR
MC5
V79
VC8TOR
Hybrid population
irs1TOR ⫻ NM3
VC8TOR ⫻ NM3
UV20TOR ⫻ NM3
UV41TOR ⫻ NM3
NM3TOR ⫻ MC5
irs1SFTOR ⫻ NM3
UV40TOR ⫻ NM3
NM3TOR ⫻ UV40
Cell line
AA8
NM3
UV40
NM3TOR
UV40TOR
Hybrid population
UV40TOR ⫻ NM3
NM3TOR ⫻ UV40
Complementation status
130
30
7.5
27
5.6
6.5
80
0.5
87
86
97
113
107
65
30
24
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫺
D37 DEB (µM)
Complementation status
5.1
1.7
1.2
1.6
1.1
1.9
2.1
⫺
⫺
⫹ Complementation, hybrids show survival response similar or near to
wildtype.
_ No complementation, hybrids show survival response similar to mutant.
All D37 values calculated from full survival curves.
Survival curves were not constructed for mutants irs1TOR, irs1SFTOR,
UV20TOR or UV41TOR. All four exhibit extreme MMC-sensitivity and it
was confirmed that survival at 20 nM MMC was ⬍1% for these cell lines.
Fig. 1. Mitomycin C survival responses of AA8, NM3, UV40 and hybrids
NM3TORxUV40, UV40TOR ⫻ NM3 and NM3TOR ⫻ 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.
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 I).
1941
J.B.Wilson et al.
Table II. Sensitivities of NM3 and UV40 to genotoxic agents and their
correction following transfection of the human FANCG cDNA
Fold sensitivity of cell line (D37 AA8/ D37
mutant or transformant)
Genotoxic
agent
D37 AA8
(wildtype)
NM3 NM3-FANCG UV40 UV40-FANCG
Mitomycin C
DEB
Melphalan
Chlorambucil
Bleomycin
Streptonigrin
Camptothecin
Etoposide
EMS
MMS
130 nM
5.1 µM
185 nM
29 nM
0.9 mU/ml
9.7 nM
41 nM
250 nM
1.5 mM
110 µM
4.3
3.0
3.0
8.8
7.5
2.2
2.2
1.5
2.7
4.6
1.2
1.3
1.9
1.4
1.6
1.1
1.2
1.2
1.5
1.4
17
4.2
9.7
10
3.6
1.9
3.0
1.7
3.1
7.3
1.3
1.4
2.1
1.3
1.3
1.3
1.9
1.2
1.6
1.5
Sensitivity of NM3 and UV40 to DNA damaging agents
Table II 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 1). 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 2A).
In contrast was the result for the radiomimetic compound
bleomycin (Figure 2B), 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.
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 II 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 2A) 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 FANCGtransformants of NM3 and UV40 with both these agents; data
not shown). The sensitivity of the NM3-FANCG and UV40FANCG transformants were similar for all agents regardless
of the differing sensitivities observed in the two mutants. For
1942
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.
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 2B and Table II).
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 3). 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
New Chinese hamster FANCG/XRCC9 mutant
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).
Table III. Frequency of spontaneous sister chromatid exchange in AA8,
NM3 and NM3-FANCG
Cell line
Number of
cells scored
Mean SCE
per cell
Standard error of
the mean
AA8
NM3
NM3-FANCG
25
25
25
11.04
12.56
12.00
3.05
2.31
2.71
(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 3, lanes 1 and 6). However, the transformants NM3FANCG and UV40-FANCG clearly show both isoforms
(Figure 3, 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 ⫻ NM3 and
NM3TOR ⫻ UV40 only expressed the FANCD2-S isoform
(data not shown).
Spontaneous levels of SCEs in NM3, AA8 and NM3-FANCG
Table III 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.
Discussion
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 II). 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 interstrand 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 doublestrand 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 II). 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 FANCGtransfectants 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 FANCD2L are corrected (Table IV). 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.
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 (5–7,54,55). 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
1943
J.B.Wilson et al.
Table IV. Cellular phenotype and expression of FANCD2-L isoform in cell lines
Cell line
Expression
of FANCD2-L
MMC
sensitivity
DEB
sensitivity
Bleomycin
sensitivity
EMS/MMS
sensitivity
Level of
spontaneous SCE
AA8 (WT)
V79 (WT)
NM3
UV40
UV40TOR ⫻ NM3 hybrid
NM3TOR ⫻ UV40 hybrid
NM3-FANCG
UV40-FANCG
⫹
⫹
⫹
⫹
R
R
S
S
S
S
R
R
R
R
S
S
S
S
R
R
R
R
S
S
nd
nd
R
R
R
R
S
S
nd
nd
R
R
Normal
Normala
Normal
Elevatedb
nd
nd
Normal
Elevatedb
aTucker
et al. (63).
et al. (18) and Liu et al. (19).
WT: wildtype.
S: hypersensitive.
R: wildtype or near-wildtype resistance.
nd: not determined.
bBusch
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 doublestrand 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 3).
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
1944
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.
Acknowledgements
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.
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Received May 29, 2001; revised August 3, 2001; accepted August 21, 2001