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1996 87: 938-948
Fanconi anemia genes act to suppress a cross-linker-inducible p53independent apoptosis pathway in lymphoblastoid cell lines
FA Kruyt, LM Dijkmans, TK van den Berg and H Joenje
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Fanconi Anemia Genes Act to Suppress a Cross-Linker-Inducible p53Independent Apoptosis Pathway in Lymphoblastoid Cell Lines
By Frank A.E. Kruyt, Lonneke M. Dijkmans, Timo K. van den Berg, and Hans Joenje
Hypersensitivityto cross-linking agents suchas mitomycin
C ("C)
ischaracteristicof cells from patients suffering
from the inherited bone marrow failure syndrome, Fanconi
anemia (FA). Here, we link MMC hypersensitivityof EpsteinBarrvirus (EBV)-immortalized FA lymphoblasts to ahigh
susceptibilityforapoptosisandp53activation.
InMMCtreated FA cells belongingto complementation group C (FAC), apoptosis followed cell cycle arrest in the G2 phase. In
stably transfected FA-C cells, plasmid-driven expression of
the wild-type cytoplasmicFAC protein relieved MMC-dependent G2 arrest and suppressed p53 activation. However, in
both FA and non-FA lymphoblasts, p53 seemed not to be
instrumentalin the induction of "C-dependent apoptosis,
sinceoverexpressionofa
dominant-negative p53 mutant
failed to affect cell survival. In addition, no differences in
the level of Bcl-2 expression, an inhibitor of apoptosis,
were
detected between FA and non-FA cellseither in the absence
or presence ofMMC. Our findings suggestthat FAC and the
other putative FA gene productsmay function in a yetto be
identified p534ndependent apoptosis pathway.
0 1996 by The American Societyof Hematology.
F
of the oxygen tensioncorrectedthis
G2 delay in primary
cell cultures'2.'' and decreasedthe rate of spontaneous chromosomal breaks in lymphocyte culture^,'^ it has been suggested that a cellular mechanism involved in detoxification
of oxygen radicals may be responsible for the FA phenoHowever, oxygen hypersensitivity seems to be a
secondary manifestation of the primary FA defect, sinceFA
fibroblasts transformed with SV40 large T antigen had lost
this feature."
DNA damage, such as that caused by MMC, UV light,
and ionizing radiation, can lead to cell cycle arrest in either
the G1 or G2 phase,
which isthoughtto facilitate DNA
repair prior to DNA replication and mitosis,respectively.
An important inducer of G1 arrest is the tumor suppressor
protein, p53 (for review, see Zambetti and LevineI7). Treatment of cells with a wide variety of DNA-damaging agents,
including MMC, is known to induce accumulation of ~ 5 3 . ' ~
AT and BS genes havebeen suggested to be involvedin the
pathway leading to p53 activation, since ionizing radiationinduced p53 accumulation was delayed or absent in AT and
BS cells.'"*'
Another function of p53 is to initiate apoptosis, which is
thoughttooccur
when cellscannotcope
with theDNA
damage and thus avoid their own survival while suffering
from an excessivemutational load. Sustained p53accumulation has been shown to induce apoptosis in a variety of cell
types."-24 However, lymphoid cellsobtained from transgenic
mice lacking p53 (p53-knockouts) were recently shown to
readily undergo apoptosis upon exposure to DNA-damaging
agents, indicating that p53-independent mechanisms can also
trigger apoptosis."
The proto-oncogene, bcl-2, a functional homolog of the
Caenorhabditis elegans gene, ced-9, has
been identified as an
inhibitor of apoptosis in mammalian ~ e l l s . ' ~Constitutive
~'~
expression of the cytoplasmic membrane protein, Bcl-2, enhanced survival by suppressing cytotoxicagent-induced
apoptosis in lymphoid cells~*-30
which appeared to be mediated through an as yet unknown mechanism downstream of
p53."
To investigate the possible role of FA genes in the pathways that control cellular survival strategies, we set out to
document the unique hypersensitivity of FA cells to MMC
in terms of p53 activation and apoptosis induction. For this
purpose,we used Epstein-Barrvirus (EBV)-immortalized
lymphoblasts derived from FA patients belonging to different complementation groups and healthy individuals. In ad-
ANCON1 ANEMIA (FA) isan autosomal recessive disease characterized by developmental abnormalities
(thumb and radius hypoplasia, microcephaly, growth delay,
and kidney abnormalities), hyperpigmentationoftheskin
(cafk-au-lait spots), and life-threatening bone marrow failure.',' In addition, FA patients have a dramatically increased
risk of developing malignancies, mainly acute myeloid leukemia and squamous cell carcinoma. Cultured FA cells exhibit an increased sensitivity to cross-linking agents such as
mitomycinC ("C)
and diepoxybutane.' Because of an
increased level of spontaneous chromosomal aberrations in
cultured cells, FA, like ataxia
telangiectasia (AT) and Bloom
syndrome (BS), is known
as a chromosomal instability disorder. In FA, cell-fusion experiments have revealed four complementation groups, A to D'; recently,a fifth group was
identified.4 In contrast to the UV-sensitivity diseases xeroderma pigmentosum, Cockayne syndrome, and
trichothiodystrophy, where disturbances in excision repair and transcription have been documented in detail,' the molecular
bases of chromosomal instability disorders arestill unknown.
The FA group C gene, FAC (according to the nomenclature recommended by Lehmann et a16), cloned by Strathdee
et al,7 is the first chromosomal instability disease gene isolated. The gene encodesa 63-kD polypeptide with no known
sequence motifs that could provide a clue to its function.
Since functionally active FAC protein apparently localizes
tothecytoplasmiccompartment
of cells,8.' the proteinis
unlikely to be directly involved in DNA repair.
Besides cross-linkerhypersensitivity, cell cyclekinetic
studies in primary fibroblasts and lymphocytes derived from
FA patients revealed a characteristic spontaneous delay and
arrestin G2."'.'' Thisphenomenonmayexplainthe
poor
proliferative properties of primary FA cells. Since reduction
From the Departments of Human Genetics and Cell Biology and
Immunology, Free University, Amsterdam, The Netherlands.
Submitted June 2, 1995; accepted September 7, 1995.
Supported by the Dutch Cancer Society.
AddressreprintrequeststoHansJoenje,PhD,Department
oj
Human Genetics, Free University, Van der Boechorststruat 7, NL1081 BT Amsterdam, The Netherlands.
The publication costsof this article were defrayed
in part by page
chargepayment. This article must thereforebeherebymarked
"advertisement" in accordunce with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-497//96/8703-00I4$3.00/0
938
Blood, Vol 87, No 3 (February l ) , 1996: pp 938-948
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939
FAGENESSUPPRESSp53-INDEPENDENTAPOPTOSIS
Table 1. Lymphoblastoid Ce
l Lines Used in the Experiments
Cell Line
GenotvDelPhenotvDe
HSC93
HSC72
HSC230
HSC536
HSC62
vu12*
VU56’
VU178’
vu202*
Wild type
FA-A
FA-B
FA-D
FA-A heterozygote
FA-A
FA-B
FA-D
References
3
50
3
3
3
4
4
4
* Cell lines are from the EUFAR cell repository (full code names
are 92VUO12-L,92VUO56-L,93VU178-L2, and 93VU202-L) and were
characterized by complementation analysis; cell line VU178-L2 has
not been reported previously.
dition, we used a complementation group C cell line that
had been corrected for MMC sensitivity by stable transfection with an episomal vector expressing wild-type FAC. We
found that FA cells are highly susceptible to ”C-,
but not
to gamma ray- or UV-, induced G2 arrest and subsequent
apoptosis. Moreover, although the FA genes appeared to
function in a pathway that suppresses MMC-dependent p53
activation, apoptosis occurred independently of p53. Thus,
FA gene products may function in pathways involved in
apoptosis and cell cycle regulation, either directly or indirectly, through DNA repair.
MATERIALS AND METHODS
Cell lines and cell treatments. FA and non-FA lymphoblastoid
cell lines used in this study (Table 1 ) were cultured at 37°C in RPM1
1640 medium (GIBCO-BRL, Gaithersburg, MD) containing 10%
newborn calf serum (Hyclone, Logan, UT) under an atmosphere of
5% CO2 in air. Stably transfected cell lines were grown in the same
medium supplemented with hygromycin (final concentration, 200
pg/mL; Boehringer, Mannheim, Germany). Cell counting was performed with a Coulter counter (Coulter Immunology, Hialeah, FL).
Treatment with MMC (Kyowa Hakko Kyoga Ltd, Japan) was
performed according to either one of two protocols: (1) exponentionally growing cells were exposed for 1 hour to MMC concentrations between 0 and 30 pmoVL, washed in Earle’s balanced salts
(GIBCO-BRL), and cultured in the absence of MMC for indicated
periods; or (2) cells in parallel cultures were grown, starting with 5
X 104/ml,in the continuous presence of low concentrations of MMC,
between 0 and 100 nmol/L as indicated, and harvested at the time
that untreated cells had undergone three cell divisions as determined
by cell counting (typically after 3 to 5 days). To assess growth
inhibition, the final cell count of untreated cultures was set at 100%.
A 6oCo source (3.4 Gy/min) was used for exposure of cells to
various dosages of gamma rays. Irradiated cells were resuspended
in fresh medium at 4 X lO’/mL and cultured for 1 or 2 days, as
indicated.
For UVC-irradiation exposure, 2 X 10‘ cells were sedimented and
resuspended in 4 mL phosphate-buffered saline (PBS) followed by
irradiation in a Pem dish (60 cm’) using a germicidal lamp (254
nm) at 10 pW/cm’, and then resuspended in culture medium.
Plasmids. pDRFAC was constructed by subcloning the 2-kb
BamHI-XbaI fragment derived from pFACC3’in the EBV-based
episomal expression vector pDR2 (Clontech Laboratories Inc, Palo
Alto, CA) containing the Rous sarcoma virus, LTR.
pDRp53m was constructed by subcloning the 1.8-kb BamHI fragment from pC53-SCX3” in pDR2. pC53-SCX3 encodes a mutated
p53proteininwhich
the valine atposition 143 is substituted for
alanine (p53-143a).
Stable transfections. QIAGEN Plasmid Kit (QIAGEN Inc, Chatsworth, CA) -purified DNA was used to produce stably transfected
cells by electroporation. Briefly, exponentially growing cells were
pelleted and resuspended inRPM1 1640 medium (GIBCO-BRL)
containing 10% normal calf serum (HyClone) at a concentration of
5 X 10‘ cells/mL. Electroporation was performed in a 4-mm cuvette
containing 800 pL cell suspension using the Easyject (Eurogentec,
Seraing, Belgium) electroporation apparatus in twin-pulse mode,
with a setting for the first pulse at 750V/25 pF/201 Ohm, and for
the second pulse at 125V/3,000pF/99 Ohm. After 24hours’ recovery
time in I O mL fresh medium, cells obtained from two independent
transfections with pDR2-based constructs were grown separately in
medium containing 200 pg/mL hygromycin for at least 3 weeks
to select for hygromycin-resistant cell populations. Since in pilot
experiments similar results were obtained in at least two independently selected stable transfectants containing the same expression
vector construct, in most experiments only one selected population
was used. In addition, parental and corresponding control cell lines
that had been stably transfected with pDR2 alone behaved similarly
in all experiments.
Apoptosisassay and cell cycleanalysis. Apoptotic cells were
assayed using propidium iodide (PI) staining and subsequent flow
cytometry analysis. The method used was essentially as described
previo~sly.~’
Briefly, 5 X 10’ cells were fixedin70% ethanol for
24 to 48 hours at -20°C. After washing with PBS, cells were permeabilized in PBS/O.I% Nonidet P-40 for 10 minutes on ice followed
by another wash in PBS. Cells were stained in PBS containing PI
50 p g / m L (Calbiochem, La Jolla, CA) and RNase A 25 pg/mL
(Sigma, St Louis, MO) for 30 minutes at room temperature. For
each measurement, lo4 cells were analyzed for fluorescence, which
was recorded by a FACScan flow cytometer (Becton Dickinson,
Mountain View, CA); cell debris was excluded onthe basis of
forward and side light-scattering properties. Cell cycle distribution,
including apoptotic cells represented by a “sub-Gi” peak, was determined using the Cellfit program (Becton Dickinson) supplied by
the manufacturer.
Immunoblotting. Cell extracts were prepared by lysis of PBSwashed cells in ice-cold sample buffer (10s cells/lO pL) containing
25 mmol/L Tris hydrochloride, pH 6.8, 1% (wthol) sodium dodecyl
sulfate (SDS), 5% (voUvol) glycerol, 2.5% 2-mercaptoethanol, and
0.25 mg/mL bromophenol blue, followed by sonification for 10 seconds on ice. After boiling for 5 minutes, cell extracts (representing
10’ cells per sample) wereloadedand electrophoresed on a 10%
SDS-polyacrylamide gel and transferred to immobulon-P membrane
(Millipore Corp, Bedford, MA). After blocking with 5% dry milk
in TBS (10 mmol/L Tris hydrochloride, pH 7.5, and 150 rnmoVL
NaCl) for l hour at room temperature, the membrane was incubated
overnight at 4°C with the antihuman p53 mouse monoclonal antibody
Do-7 (Dako, Glostrups, Denmark) or antihuman bcl-2 oncoprotein
mouse monoclonal antibody 124 (Dako), diluted 1:3,000 in TBS
containing I % bovine serum albumin ([BSA], Sigma). Subsequently,
after washing four times in TBST (TBS supplemented with 0.05%
Tween 20; Sigma), the membrane was incubated for 1 hourwith
biotinylated F(ab‘), fragments of rabbit antimouse IGgs (Dako) diluted 1:3,000 in TBSBSA, washed three times in TBST, and incubated with a 1:3,000 dilution of peroxidase-conjugated streptavidin
(Dako) in TBSlBSA for 1 hour; all steps were performed at 4°C.
Blots were developed by enhanced chemiluminescence according to
the instructions of the manufacturer (Amersham, Arlington Heights,
IL).
Quantification of p53 levels was performed by densitometry using
a Charge Coupled Device camera and a Cybertech Image Processing
system (Cybertech, Berlin, Germany).
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940
K R U M ET AL
A
o L
1
10
I
100
MMC (nM)
B
1
10
100
MMC (nU)
Fig 1. FAC
suppresses
MMC-induced apoptosis in FA-C
lymphoblasts. (A) HSC536 cellswere stably transfected with thecontrol vector pDR2 lHSC536P; 0 ) or the seme vector containing FAC
cDNA lHSC536FAC; 0 ) and assayed for growth inhibition by MMC;
IC, values were 4.5 and 60 nmol/L, respectively. The number of cells
in untreated cultures was set at 100%. IBI In the same experiment,
the fraction of apoptotic cells was determined following PI staining
and FACS
analysis.
Percentages
are corrected for background
apoptosis present in cultures grown in the absence of MMC, which
was on the order of 5%.
RESULTS
FAC suppresses MMC-induced apoptosis in FA-C cells.
To assess whether apoptosis is involved in the hypersensitivity of FA cells to the growth-inhibitory effect of MMC, we
investigated MMC-induced apoptosis inFA lymphoblasts
belonging to complementation group C and in the same cells
expressing FAC. For this purpose, we used HSCS36 cells,
which have been shown to express a defective FAC protein
due to substitution of a leucine for a proline at position SS4.33
We generated stably transfected HSC536 cells containing
either the episomal EBV-based expression vector pDR2
alone or pDR2 inserted with the cDNA encoding wild-type
FAC protein. In growth-inhibition assays, the wild-type
FAC-expressing cell line HSCS36FAC was greater than 1
order of magnitude more resistant to MMC than the control
cell line HSCS36P (Fig IA). In the same experiment,
apoptosis was determined by flow cytometry of PI-stained
permeabilized cells. In this assay, apoptotic cells show up
in a sub-G1 peak, since chromatin condensation and DNA
cleavage, which is characteristic of apoptotic cells, reduces
the PI fluorescence intensity to a level less thanthat of
diploid cell^.'^^^^ Quantification of the fraction of apoptotic
cells in untreated cell cultures showed a background level
of less than 5% in bothcell lines. However, uponMMC
exposure in HSC536P cells, the fraction of apoptotic cells
started to increase at 3 nmol/L MMC and continued to increase in a dose-dependent way, whereas in the corrected
cell line apoptosis was not observed at concentrations less
than 30 nmoVL. Fluorescence microscopy and determination
of internucleosomal DNA cleavage by agarose gel electrophoresis confirmed that the occurrence of the sub-G1 peak
correlated with the occurrence of cells displaying subnuclear
bodies and DNA-cleavage patterns characteristic of apoptotic cells (not shown).
These findings demonstrate that hypersensitivity of
HSCS36 cells to MMC is correlated with excessive induction
of apoptosis, which is suppressed by expression of wild-type
FAC.
MMC-induced apoptosis in lymphoblasts is preceded by
G2 arrest. The cellular response to chronic low-dose MMC,
such as applied in Fig 1, is compound, since it reflects the
cellular recovery from cytotoxic lesions and the response to
new lesions being continuously introduced. Application of
a pulse treatment with higher-dose MMCwouldsimplify
interpretation of the data, since only the recovery response
would be observed. To determine whether the latter protocol
could beused
to distinguish FA from non-FA cells in
apoptosis assays, HSCS36PandHSCS36FAC
cells were
treated with various high concentrations of MMC ( 1 to 30
pmol/L) and MMC-dependent apoptosis was examined after
3 days' recovery time. As observed earlier in these cell lines,
the background level of apoptotic cells was again approximately S% (Fig 2A). In HSCS36P cells treated with 1 pmol/
L MMC, approximately 15% of the cell population became
apoptotic, whereas in FAC-corrected cells IO pmol/L MMC
was required to induce a similar level of apoptosis. Up to
10 p m o K MMC, a dose-dependent increase in the fraction
of apoptotic cells was observed in HSCS36P cells, whereas
at 30 pmoVL MMC, both cell lines exhibited similar levels
of apoptosis (-60%), indicating that this high concentration
no longer distinguishes a FA from a non-FA phenotype. In
addition, MMC treatment appeared to induce a small population of polyploid cells (>G2) in HSCS36P cells, whereas in
FAC-corrected cells, whichhadan
elevated background
level of polyploid cells, MMC exposure had no such effect
(Fig 2B).
The effect of MMC treatment on cell cycle distribution
suggested that the onset of apoptosis was preceded by a
sharp decrease of the G1 cell fraction with a simultaneous
accumulation in the G2 phase (Fig 2A).To examine these
phenomena in more detail, time course experiments were
performed. Cells were treated with I , IO, and 15 pmol/L
MMC for 1 hour and analyzedafter 8, 24, 48, and 72
hours' culture. Quantified cell cycle fractions are shown
graphically in Fig 2B; during this time course, cell cycle
fractions remained constant in the absence of MMC (not
shown).
Within 24 hours, treatment with 1 pmol/L MMC resulted
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941
FA GENES SUPPRESS p534NDEPENDENT APOPTOSIS
A
HSC536P
fl
HSC536FAC
l,
,3%
HSC536FAC
1
MMC(pM)
-0-
sub G 1
-0-
G1
I
5%
6o
50
1
0
30
-O-
0
f]
]
17%
4%
1
HSC536P
HSC536FAC
10 pM
10 p M
70
60
fl
"l
l
29%
s u b G1
-0-
sub G 1
-0-
G1
I
5%
2.5
O
I
81
8
0
l 2 3 26 1
6048
72
0
l2
24
36
48
60
72
35%
5
l0
-0-
I
HSC536FAC
15 )rM
HSC536P
15 V M
-_L1
10
0
time (h)
"l
24
16
48
60
72
time (h)
30
"l
0
l2
1
W
o
4d0 - 4
PI fluorescence
Fig 2. MMC-induced cell cycle changes and apoptosis in FA-C lymphoblasts and genetically corrected counterparts. (AI HSC536P and
HSC536FAC cells were exposed t o the indicated concentrations of MMC for 1 hour and assayed for apoptosis after 3 days' culture in fresh
medium. PI-stained cells were analyzed by flowcytometry, allowing detection of apoptotic cells as sub-G1 material, indicated by horizontal
bars. Percentages of apoptotic cells for each sample are indicated. (B) Time course for the effect of MMC on cellcycle distribution. HSC536P
and HSC536FAC cells treated with 1, 10, or 15 pmollL MMC for1 hour were fixed after the periodsindicated. PI-stained calls were analyzed
by flowcytometry, and percentages of cells present in the different phases of the cell cycle were quantified.
in a sharp decrease in the fraction of HSC536P cells in G1
and a simultaneous accumulation in G2 (-70%). At later
time points, the fraction of cells in G1 and G2 became equal,
but the absolute number of cells in each fraction was reduced
10% to 15% as a result of many cells having undergone
apoptosis. Thus, upon treatment with l pmol/L MMC,
HSC536P cells progress from G1 to G2, and following accumulation in G2, either partially transit to G1 or exit the cell
cycle to undergo apoptosis. At 10 and 15 pmol/L MMC and
24 hours posttreatment, the majority of these cells accumulated in G2, whereas apparently part of the cells underwent
apoptosis directly from G1, as illustrated by the approximately 10% increase of the sub-G1 fraction and a similar
reduction of G2-arrested cells, as compared with the situation
found at 1 pmol/L MMC treatment. Whether G2-arrested
cells undergo apoptosis directly or following transition to
the G1 phase cannot be inferred from this experiment.
In HSC536FAC cells, 1 pmol/L MMC hardly affected the
cell cycle distribution; after 24 hours, a small decrease in
G1 and an increase in G2 were observed, which returned to
normal values at 48 hours posttreatment. In contrast to the
situation in HSC536P cells, massive apoptosis induction at
10 and 15 p m o E MMC in HSC536FAC cells was still
accompanied by a fraction of cells (20% to 30% after 72
hours) successfully completing mitosis. Furthermore, in both
cell lines, MMC treatment appeared to stimulate progression
from G1 to G2, as demonstrated by the increase in S-phase
cells 8 hours posttreatment.
In conclusion, wild-type FAC expression in HSC536 cells
reduces MMC-induced accumulation in G2 and suppresses
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942
the onset of apoptosis, as well as progression to polyploidy,
in this cell fraction.
High-susceptibility for MMC-induced apoptosis is common to FA cells and spec@ for MMC. To examine whether
hypersensitivity to low-dose "C-induced
apoptosis is a
common characteristic of the FA cellular phenotype, we determined MMC-induced apoptosis in FA lymphoblasts representing complementation groups A, B, C, and D. As controls, we used a normal human and a FA-A heterozygous
cell line (Table 1). Cells were pulse-treated with MMC and
assayed for apoptosis after 3 days. In the cell lines, background apoptotic levels were not increased following treatment with 0.1 p m o K MMC (Fig 3A). However, in all FA
cell lines studied, approximately 10% of the cells became
apoptotic after treatment with 1 p m o K MMC, whereas a
10-fold-higher concentration was required to induce a similar level of apoptosis in control lymphoblasts. In addition,
PI fluorescence histograms for these cell lines were similar
to those depicted inFig 2A, with histograms for FAand
wild-type cell lines being similar to those for HSC536P and
HSC536FAC cells, respectively (results not shown). Cultures of cells with wild-type MMC sensitivity were appreciably affected in cell cycle distribution only at the two highest
MMC concentrations. However, the elevated level of polyploid cells found inuntreated HSC536FAC cells wasnot
observed in wild-type cell lines (results not shown).
Next, we tested whether defective FA genes also affect
the apoptotic response to gamma ray or UV irradiation, for
which hypersensitivity has been demonstrated in AT and XP
cells, respectively. FA and non-FA lymphoblasts, including
HSC536-derived transfectants, were gamma- or UVirradiated and the level of apoptosis wasassayedat 1 or
2 days posttreatment. Both gamma ray and UV irradiation
induced a dose-dependent increase in the fraction of apoptotic cells (Fig 3B and C). Except for some variation in the
level of apoptosis found at fixed doses of gamma irradiation,
no differences in apoptotic response were observed between
FA and non-FA cells (Fig 3B).The FA phenotype apparently
also didnot interfere with the apoptotic response toUV
irradiation, as depicted for HSC536-derived transfectants
(Fig 3C). Thus, the specific hypersensitivity of FA cells to
cross-linking agents extends to apoptosis.
FA genes suppress MMC-induced p53 activation and do
not affect Bcl-2 expression. To investigate the molecular
mechanism leading to apoptosis in normal
and
FA
lymphoblasts, we focused on the expression of two predominant modulators of cell death, p53 and Bcl-2.
First, we examined p53 expression by immunoblotting in
normal lymphoblasts and a panel of FA cell lines representing complementation groups A to D, including HSC536P
and HSC536FAC cells, following continuous exposure to
various low concentrations of MMC. Interestingly, as shown
by representative experiments in Fig 4A and C, treatment
with 3 or 10 nmoVL MMC resulted in a clear increase in
p53 expression in all FA lymphoblasts examined except for
HSC230 (FA-B) cells, which exhibited only a small increase
in p53 levels, whereas at least 10-fold-higher concentrations
were required to induce p53 expression in cell lines with
wild-type MMC sensitivity. The weak p53 response in
HSC230 cells appeared to be characteristic for the FA-B
KRUYT ET AL
-0-
HSC93
-v-
vu12
-A-
HSC72
- 0-
VU56
-V-
HSC230
- @ -
0.1
l
10
-0-
HSC62
-m-
vu202
-0-
HSC93
-"
vu12
-0-
HSC536P
L
-0-
0
2
4
6
7 -irradiation
8
1
HSC536
HSC536FAC
0
(Gy)
C
60
1
A
5
50
-
40
U)
Q,
0
.-"0
Sn
30
20
I
: 10
a
0
0
5
10
15
20
UV (J/m')
Fig 3. Comparison of apoptosis induction by MMC and gamma and
UV radiation in FA and non-FA lymphoblasts. (A) Wild-type and FA
lymphoblasts, representing complementation groups A, B, C, and D,
were treated with different concentrations of MMC for 1 hour and
incubated in fresh medium. After 3 days fractions of apoptotic cells
were quantified in PI-stained cells. (B and C) Cell lines were a x p o d
to different dosages of gamma or UV irradiation and, following
growth for 2 days in fresh medium, were assayed for apoptais.
Percentages of apoptotic cells are corrected for background levels
found in untreated cell cultures, which varied between 2% and 5%.
subtype, since VU178 showed a similar response (not
shown). The observation that HSC536FAC cells accumulate
p53 only after treatment with 100 nmoVL MMC, resembling
the situation found in wild-type HSC93 and VU12 cells,
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TOSIS p53-INDEPENDENT
FA GENES SUPPRESS
MMC (nM)
A
943
0 3 10 30 100
HSC93
"
V
C
vu12
HSC536P
HSC536FAC
m
v)
n
"
-A-
HSC93
-
HSC536P
0-
- e-
HSC536FAC
-
A-
HSC72
-
0-
HSC230
HSC72
0
HSC230
"
"
C
-
MMC @M)
B
0
"-
l 2.5 5
HSC62
10 30
P53
vu12
0
20
40
60
80
100
MMC (nM)
Fig 4. FA genes affect MMC-induced p53 response. (A) Cell lines
were continuously exposed to various concentrations of MMC and
harvested after 3population-doublings had occurred in untreated
cultures (3 to 5 days). Cell extracts, lo5 cell equivalents per lane,
were loaded and electrophoresed on SDS-PAA gels, whereafter p53
expression was determined by immunoblotting. (B) p53 andBcl-2
expression in the indicated cell lines, treated for 1 hour with various
concentrations MMC andharvested after 24 hours.Levels of p53
and Bcl-2 expression were determined simultaneously in the same
extract. (C) Graphic representationof p53 levels shown in (A), relative
to levels found in untreated cells.
Bd-2
P53
-"
VU56
"
BcI-2
P53
"
"
vu202
Bcl-2
-
-
."
-
whereas in the FA control cell line HSC536P p53 activation
was readily detected following treatment with 3 nmol/L
MMC, shows that FAC suppresses MMC-induced p53 activation.
Next,we
investigated p53 and Bcl-2 expression in
lymphoblasts recovering from high-dose MMC treatment,
ie, 1 hour of treatment with 1 to 30 pmol/L MMC. Time
course experiments in these cells showed that p53 accumulated within 8 hours after MMC treatment, reaching a maximum at 24 hours, followed by a decrease at 48 hours (not
shown). Figure 4B shows p53 accumulation in lymphoblasts
24 hours posttreatment. A small increase in p53 expression
was detected in wild-type VU12 cells exposed to 2.5 and 5
pmol/L MMC, whereas treatment with IO and 30 pmoVL
MMC strongly induced p53 accumulation. In contrast, 1
pmoVL MMCwas sufficient to induce strong p53 accumulation in FA cell lines VU56 and VU202. These findings were
confirmed in other wild-type and FA cell lines butnotin
FA-B cells, which again showed only a weak p53 response
(not shown). In addition, similar Bcl-2 expression levels
were detected in wild-type and FA cells, whichwasnot
affected by MMC treatment (Fig 4B). This indicates that
hypersensitivity of FA cells to MMC-induced apoptosis is
not due to an altered expression level of the cell-death inhibitor, Bcl-2.
Taken together, these results show that MMC-inducedp53
accumulation is suppressed by FA genes, although this was
much less obvious in the case of FA-B cells. Moreover,FAC
appears to function in a cytoplasmic pathway that by an asyet-unknown mechanism antagonizes MMC-dependent p53
accumulation.
Gamma or UV irradiation-induced p53 activation is not
aflected by FAC. To investigate whether FAC might also be
involved in pathways leading to gamma ray- or UV-induced
p53 accumulation, we studied p53 activation in HSC536P
and HSC536FAC cells. Cells were exposed to various dosages of gamma or UV radiation, whereafter p53 induction
wasmonitored.As depicted in Fig5A and B, essentially
no dose-dependent differences in p53 accumulation were
detected in these cell lines, although UV-induced p53 accumulation seemed to reach higher levels in HSC536P as comparedwith HSC536FAC cells. Similar observations were
made in other FA and non-FA cells treated with both types
of radiation (not shown). In addition, as observed in MMCtreated lymphoblasts, Bcl-2 expression was not affected by
either gamma or UV irradiation in these cells.
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
KRUYT ET AL
944
HSC536P
HSC536FAC
A
y
(
0 1
2.5
5
W
1 0 0 1 2.5
5
10
"
"
P53
Bcl-2
0
U V (Jim')
1 5 1 0 2 0 0 1 5 1 0 2 0
""""om
P53
"""""
Bcl-2
B
-0-
HSC536P
-0-
HSC536FAC
-0-
HSC536P
-0-
HSC536FAC
v
0
-
2
4
6
8
1
0
0
0
5
10
15
20
UV (J/m')
Fig 5. (A) p53 response induced bygamma or UV irradiation in FA
and non-FA cells. p53 and Bcl-2 expression levels were determined
by immunoblotting 2 or 3 hours after gamma or UV irradiation, respectively, in HSC536P and HSC536FAC cells. Quantified p53 levels
are shown graphically in (B), depicted relative to the level found in
untreated cells.
These findings indicate that FA gene products do not function in the pathway leading to gamma ray- or UV-induced
p53 accumulation.
M r c h r i s m of npoptosis i n d ~ r c t i o i~n~ FA nrzd non-FA
!\.r~l~Jlzohl~~.sts
i s p53-inclrperzd~~rrr.
Accumulation of p53 is
known to be associated with DNA damage and to mediate
GI/S cell cycle arrest. which may facilitate DNA repair. On
the other hand. when a cell cannot cope with the genomic
insult, p53 can inducecell death. Our resultsindicate that
functional FA gene products can suppress induction of p53,
since in FA lymphoblasts p53 accumulation is detected at
MMC concentrations that do not induce p53 expression in
wild-type cells. In addition. expression of wild-type FAC in
HSC536 cells repressed p53 activation. lending to a MMC
concentration-dependent accumulation ofp53 similarto that
the increase in
seen in wild-typelymphoblasts.Although
p53 expression appeared to correlate with the occurrence of
apoptotic cells. MMC-induced cell cycle arrest at the GI/S
borderwas not apparent in the lymphoblastoidcelllines
studied (Fig 28). This suggests that p53 does not properly
exert its GI/S checkpoint function in these EBV-transformed
lymphoblasts. even though i t still might be involved in mediatingapoptosis. However, the weakMMC-dependent p53
responsefound in FA-Bcellsdoes
not reflect the strong
induction of apoptosis (Figs 3Aand4Aand
C), and may
suggest that other mechanisms could be involved in mediating apoptosis. Moreover. it is questionable whether p53 can
exert its proper function in EBV-transformed lymphoblasts.
Analogous to the situation found in SV40-immortalized fibroblasts. where the function ofp53 is abrogated by complex
formation with the oncogene protein large T antigen.75."'p53
function in EBV-transformed lymphoblasts may be impaired
by EBV-encoded proteins EBNA-5 and BZLFI, which have
been reported to interact with p53.7'.'' In fact,BZLFI has
been shown to inhibitp53-dependenttransactivation
in
lymphoid cells."
To investigate whether p53 mediates MMC-induced
apoptosis in EBV-transformed FA and non-FA lymphoblasts.
we stably transfected a panel of cell lines with the expression
vector pDR2 containing cDNA encoding the dominant-negative-acting mutant p53- 143al a. and examined MMC-dependent growth inhibition and apoptosis. Among other defects in
function. this m ~ ~ t a form
n t of p53 has lost its ability t o suppress
cell growth and fails to either activate or repress transcription,
and in addition. is thought to inactivate wild-type p53 by heterooligomer formation."
In immunoblotting experiments (Fig6). we found a threefold to sixfoldoverexpression
of p53-143aIa in stably
transfected cells as compared with the level of p53 detected
in control cell linestransfected with the emptyexpression
vector. as determined by densitometry. If p53 would mediate
MMC-induced apoptosis in EBV-transformed lymphoblasts.
expression of mutant p53 in FA lymphoblasts would be expected to enhance their MMC resistance. However, as depicted for HSC93, HSC536. and VU56 cells (Fig 7A and
B), p53-I43ala-expressing transfectants of both normal and
FA cells did not behave differently in either growth-inhibition or apoptosis assays. indicating that wild-type p53 function is not essential for MMC-induced apoptosis in lymphoblastoid cells.
DISCUSSION
In this study. we show that hypersensitivity of EBV-immortalized FA lymphoblasts to the growth inhibitory effect
of low-dose MMC is due to an excessive accumulation in the
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
945
FA GENESSUPPRESSp53-INDEPENDENTAPOPTOSIS
G2 phase of the cell cycle that precedes massive induction of
apoptosis. As illustrated by restored expression of wild-type
FAC in stably transfected FA-C lymphoblasts, this protein
suppressed both low-dose MMC-induced G2 accumulation
I and2). At high-dose MMC pulse
andapoptosis(Figs
treatment (30 pmolk),similar levels of apoptotic cells were
detected in both FA-C and FAC-corrected stably transfected
cell cultures. Time course experiments showed that MMCinduced G2-accumulated cells eithertransit to GI or exit the
cell cycle to undergo apoptosis. The percentage of cells in
GI was inverselycorrelated with the MMC dose. Already
at low-dose MMC, only a minor proportion of FA-C cells
were in G I , accompanied by a strong increase in the level
of apoptotic cells, in contrast to FAC-corrected cells, which
showed a similar response only at an approximately IO-fold
higher MMC concentration. The time course experiments do
not allow us to conclude whether the cells undergo apoptosis
directly from the G2-arrested state or shortly after transition
to G I . Furthermore, time course studies showed MMC-induced accelerated GI/S transition in lymphoblasts, as indicated by an increase in S-phase cells 8 hours posttreatment
high sensitivity to
(Fig2B). In primaryFAlymphocytes.
MMC-induced G2 delay and arrest within this compartment
has been reported previously."' but accelerated GI/S transition was not observed. Therefore, our finding in EBV-transformed cells may be related to the presence of virally encoded proteins.
Spontaneous G2 delay observed in primary FA cells has
been interpreted to reflect impaired DNA repair. However,
under reduced oxygen tensionthischaracteristicis
lost.
which has led to the hypothesis that FA cells are defectivein
a defense mechanism involved in detoxification of activated
oxygen species.".l5 In contrast to primary FA fibroblasts and
lymphocytes, FA fibroblasts transformed with SV40 large T
antigen no longer exhibit oxygen hypersensitivity. However,
since SV40-transformed fibroblasts still show MMC hypersensitivity. oxygen sensitivity seems to reflect a secondary
consequence of the primary defect in FA."' We have found
oxygen hypersensitivity also to be absent in FA lymphocytes
upon EBV immortalization (H. Joenje and F.A.E. Kruyt,
1995): moreover, in thepresent
unpublishedresults,May
study, we failed to observe significant differences in G2 cell
fractions betweenuntreated FA and non-FA cells(Fig 2
and results not shown), which is in contrast to the reported
spontaneousG2delay in nontransformedFA cellsand is
also likely to be a result of EBV immortalization.
Despite the above differences between primary and virustransformed FA cells, both cell types are hypersensitive to
cross-linkers.'" We found high susceptibility to MMC-dependent apoptosis to be a novel characteristic of
FA
lymphoblasts. In FA and non-FA cells, no differences were
observed in the apoptotic responsetriggered
by either
gamma or UV irradiation (Fig 3B and C), treatmentsknown
to evoke hypersensitivity in AT and XP cells, respectively.
This substantiates the notion that the genes defective in AT,
XP, and FA. although all involved in maintaining genomic
stability, clearly function in separate pathways.
Possible jirnctior1.s of FA gene proriucts in tlw repression
of MMC-induced p53 crcclrmlrlrltion. Ionizing radiation and
many cytotoxic drugs, including MMC, are known to induce
1
p53/p53m
-
2
3
4
-
-
5
6
Fig 6. p53 and p53-143ala expression in control and mutant p53
stably transfected cell lines asdetermined by immunoblotting. Lanes
1, 3, and 5: pDR2 stably transfected control cell lines derived from
HSC93,HSC536, and VU056, respectively;lanes 2, 4, and 6 their
mutant p53-expressing counterparts.
accumulation of the tumor suppressor protein, p53. which
typically leads to cell cycle arrest at the GI/S border, thus
allowing DNA repair. The precise nature of signals that activate or inactivate p53 are presently unknown. although DNA
damage is known to have a triggering function.
In this study, wefound FA cells, representing complementation groups A. C, and D, to be highly susceptible to MMCdependent p53 accumulation, ie, accumulation was detected
atapproximately
IO-fold-lower MMC concentrations as
compared with wild-type cells (Fig4). In FA-B cells, we
detected only a weak p53 response following MMC treatment, suggesting that FA-B cells are different in this respect
from the other FA subtypes. Restored wild-type FAC expression in FA-C cells essentially normalized MMC-dependent
p53 accumulation, whereas no such effect was detected following gamma or UV irradiation, although FAC expression
seemed to reduce the level of UV-dependent p53 induction
to some extent (Fig S). These results indicate that FA gene
productsspecificallyact
tosuppress p53activation in response to MMC.
Several mechanisms can be considered to explain the differences in p53 response,whereby FA geneproducts may
function in a feedback mechanism that suppresses p53 activation. The notion that FA gene products are directly involved in DNArepair4' has been contradicted for FAC.
which is localized to the cytoplasm. Moreover. FAC appears
to be functionally active only in this cellular compartment,
since a nuclear-directedfusionprotein,containing
FAC
linked to the nuclear localization domain of the SV40 large
T antigen, is unable to correct for MMC hypersensitivity in
FA-C cells, whereas this potential is restored by mutational
inactivation of the nuclear localization domain (H. Youssoufian, personal communication, March 1995). Therefore. two
main alternatives may be suggested for FA gene function:
( I ) a nuclear target, eg, DNA, provides a signal for FA gene
products to be activated, which induce an unknown cascade
leading to activation of specificfactors counteracting the
cytotoxiceffects of cross-linking agents: or (2) FA gene
products operate in a cytoplasmic system designed to detect
cross-linkers, leading to avoidance of cross-link damage.
The first possibilitywouldrequire
"inside-out" signal
transduction,whereby
a nuclearsignal activates a cytoplasmicpathway, a mechanism that has been considered
unlikely?'Alternatively,DNAfragmentsoriginating
from
DNA repairactivity may leak to thecytoplasm, thereby
forming a signal for FA gene products to become activated.
On the other hand, analogous to UV-induced signal transduc-
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946
K R U M ET AL
tion pathways, involving the detection ofUV light at the
cell membrane via an unknown receptor, leading to tyrosine
kinase-mediated activation of immediate-early genes such
as c-fos and c-jun (for review see Blattner et a143),a similar
type of cascade might be involved in the cellular response
to cross-linking agents. As in the case of the UV response,
which is still functional in enucleated cells, indicating that
a nuclear signal is not required:’ FAC may function in a
MMC-dependent signal transduction pathway. Thus, cells
may possess an extranuclear “cross-linker receptor” which
is activated by cross-linkers and induces a response that
counteracts the cytotoxic effects of cross-linking agents. FA
gene products may either function in the detection of crosslinkers or be active in a signal transduction cascade induced
by the activated cross-linker receptor, including enzymes
that counteract cross-linker cytotoxicity such as specific
DNA-repair proteins. Alternatively, these enzymes may
function to detoxify cross-linking agents in the cytoplasmic
compartment, as suggested by Youss~ufian.~
During evolution, a cytoplasmic defense system may have evolved that
is specifically designed to protect DNA from naturally occurring cross-linkers, which are of both endogenous and exogenous origin (Joenje and Gillels and references therein);
examples of endogenously produced cross-linker agents in-
A
-4-
HSC93P
-A-
HSC93p53m
-0-
HSC536P
40
-0-
HSC536p53m
20
-0-
100
h
80
S
Y
5
2
60
0)
VU56P
-.-
0
VU56p53m
1
10
100
MMC (nM)
40
I
I
1
10
100
MMC (nM)
Fig 7. MMC sensitivity of FA and wild-type lymphoblasts is unaffected by overexpression of a dominant-negative p53 mutant. MMCinduced growth inhibition (A) and apoptosis (B) of wild-type (HSC93)
and FA (HSC536 and VU561 lymphoblasts stably transfected with a
vector expressing the dominant-negative mutant p53-143ala.
clude singlet oxygen and several dialdehydes resulting from
lipid peroxidation.
Regardless of the underlying mechanisms ofFA gene
function, the putative FAB gene product apparently functions differently from other FA gene products, as indicated
by the atypical p53 response observed in FA-B cells. This
function might involve a more direct role in cell cycle regulation. Recently, Digweed et ala showed that overexpression
of a small cyclin-related protein, designated SPHAR (for Sphase response), partially corrected the permanent repression
of DNA synthesis observed in cross-linker-treated primary
FA-A fibroblasts. In addition, in untreated stably transfected
HSCS36FAC cells, we found an increased proportion of
polyploid cells as compared with the control transfectant
HSCS36Pand other nontransfected lymphoblast cell lines
(Fig 2). This cell fraction likely represents polyploid cells
resulting from endomitosis, a process in which a new round
of DNA replication occurs before mitosis. Endomitosis has
been a frequent finding in primary FA lymphocyte cultures,”
as well as in non-FA lymphocytes exposed to high levels of
oxygen.4sWe are currently exploring the possibility that
FAC might be directly involved in G2h4 transition.
Rosselli et aI4‘ recently reported that FA lymphoblasts are
defective in both MMC- and gamma-radiation-induced p53
accumulation. These results are in contrast to our data. The
discrepancy might be explained by their use of a control cell
line with an unusual p53 response. For example, short-term
exposure to low-dose MMC (10 and 100 ng/mL equivalent
to 30 and 300 nmol/L MMC) for, at most, 16 hours failed
to induce p53 accumulation in FA-C and FA-D cell lines,
but did activate p53 in their control cell line. In our hands,
24-hour exposure to 100 nmol/L MMC did not induce p53
accumulation in bothFAandnon-FA
cells, including
HSC536P and HSCS36FAC cells (not shown), suggesting
that their control cell line was somehow hyperresponsive.
The discrepancies concerning ionizing radiation-induced
p53 activation might be explained in a similar way. Moreover, in our hands, the short-term low-dose protocolused
by Rosselli et a1 is not suitable to demonstrate specific crosslinker hypersensitivity of FA cells. Instead, we used protocols that clearly distinguish FA from non-FA cells, ie,
chronic low-dose MMC treatment ( 5100 nmol/L) or 1 hour
of exposure to high MMC concentrations ( 1 to 30 pmoVL,
pulse treatment), which revealed clear differences in p53
activation (Figs 4 and 5). Furthermore, we observed some
variation in the sensitivity of FA and non-FAcells to gamma
radiation-induced apoptosis, which was not related to the
FA phenotype, suggesting that the somewhat enhanced resistance of the two FA cell lines to gamma-radiation-induced
apoptosis as observed by Rosselli et a1 maybe related to
such variations.
Role of FA genes in the suppression of p53-independent
apoptosis. Despite the MMC-dependent accumulation of
p53, G1 arrest was not apparent in the lymphoblastoid cell
lines used, as illustrated by cell cycle kinetic studies in the
HSC536-derived stable transfectants (Fig 2). Furthermore,
in FA-B cells, the weak MMC-dependent p53 response did
not reflect the strong induction of apoptosis (Figs 3A and
4A). Since it has been reported that p53 function is impaired
by interaction with the EBV-encoded proteins EBNA-S and
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
FA GENES SUPPRESS p53-INDEPENDENT APOPTOSIS
BZLF1,37338
these findings suggested that p53-independent
mechanisms were mediating apoptosis. Indeed, overexpression of the dominant-negative mutant, p53-143ala,
known to be impaired in both transactivation and transrepressor function (reviewed in Zambetti and Levine17),failed to
affect MMC-dependent cell survival and apoptosis (Fig 7).
This finding is in agreement with the results of Strasser et
al>5 who showed apoptosis to be induced in MMC-treated
lymphoid cells derived from p53-knockout mice. Although
wild-type FA genes can act to prevent or avoid MMC-induced and p53-independent apoptosis in lymphoblasts, it remains possible that in primary FA cells elevated p53 levels
will both mediate G1/S arrest and contribute to the induction
of apoptosis. Whether FA gene products only counteract the
cytotoxic effects of MMC at low concentrations, thereby
preventing apoptosis, or are also linked directly to p53-independent apoptotic pathways remains to be elucidated. The
latter possibility is especially attractive, since it would help
to explain a number of clinical symptoms in FA. For example, FA patients typically suffer from bone marrow failure
due to impaired hematopoiesis. Since cytokine-dependent
repression and induction of apoptosis is known to play an
important role in the maturation of cells belonging to the
hematopoietic and immune system (for reviews, see Sachs
and L ~ t e m
and
~ Allen
~
et a148), bone marrow failure in FA
may be related to an exaggerated tendency of hematopoietic
progenitor cells to undergo apoptosis, which would implicate
wild-type FA genes in hematopoietic-tissue homeostasis
based on apoptotic mechanisms. Evidence for such a mechanism was recently provided by Segal et al,49 who demonstrated that normal hematopoietic progenitor cells exposed
to antisense oligonucleotides complementary to FAC mRNA
had inhibition of growth. In addition, in the present study,
no aberrant expression levels of the apoptosis inhibitor Bcl2 were found in FA lymphoblasts; Bcl-2 is known to oppose
apoptosis in hematopoietic cells. Thus, FA genes might have
a specific function to suppress apoptosis independently of
Bcl-2.
ACKNOWLEDGMENT
We thank A.G. Jochemsen for the plasmid pC53-SCX3 and for
comments on the manuscript, F. Arwert for critically reading the
manuscript, and Q. Waisfisz for helpful discussions.
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