Hum Genet (1997) 101 : 121–125
© Springer-Verlag 1997
O R I G I N A L I N V E S T I G AT I O N
Charles E. Ogburn · Junko Oshima · Martin Poot ·
Ru Chen · Kristin E. Hunt · Katherine A. Gollahon ·
Peter S. Rabinovitch · George M. Martin
An apoptosis-inducing genotoxin differentiates heterozygotic carriers
for Werner helicase mutations from wild-type
and homozygous mutants
Received: 21 April 1997 / Accepted: 25 July 1997
Abstract Immortalized B lymphocytes from Werner syndrome subjects are shown to be hypersensitive to 4-nitroquinoline-1-oxide (4NQO), supporting earlier work on T
lymphocytes. We also show that B cell lines from clinically normal heterozygous carriers exhibit sensitivities to
this genotoxic agent, which are intermediate to those of
wild-type and homozygous mutants. 4NQO is shown to
induce an apoptotic response. These data encourage research on DNA repair with such cell lines and raise the
question of an enhanced sensitivity of the relatively
prevalent heterozygous carriers to certain environmental
genotoxic agents.
Introduction
The Werner syndrome (WS) is a rare segmental progeroid
syndrome (Martin 1978) caused by mutations in a member (WRN) of the RecQ family of helicases (Yu et al.
1996). The phenotype has been described as a “caricature
of aging” (Epstein et al. 1965). Patients typically die prematurely of either a myocardial infarction or cancer (Epstein et al. 1966). Preliminary evidence has suggested that
heterozygous carriers are at increased risk of cancer (Goto
et al. 1981) and that a polymorphic form of WRN may influence one’s risk of developing myocardial infarction (Ye
et al. 1997).
While the molecular basis for the WRN mutation differs among our families and there is compound heterozygosity in two of them, most or all of the mutations so far
identified via the clinical diagnosis of WS (Yu et al. 1996,
1997; Oshima et al. 1996) may be null mutations since not
a single missense mutation has so far been identified. A
reasonable assumption, therefore, is that the levels of the
C. E. Ogburn · J. Oshima · M. Poot · R. Chen · K. E. Hunt ·
K. A. Gollahon · P. S. Rabinovitch · G. M. Martin (Y)
Department of Pathology, Box 357470, 1959 N.E. Pacific Avenue,
Health Sciences Building, Seattle, WA 98195, USA
Tel.: +1-206-543-5088; Fax: +1-206-685-8356;
e-mail: gmmartin@u.washington.edu
putative Werner helicase activity in heterozygous carriers
is approximately 50% of wild type. Since the prevalence
of the carriers is as high as 1/150 to 1/200 (Yu et al. 1996),
a deleterious phenotype associated with the carrier state
could be of potential public health concern.
Materials and methods
Table 1 lists the lymphoblastoid cell lines used for this study. “Immortalization” of peripheral blood B lymphocytes with EpsteinBarr virus utilized method 2 of Wall et al. (1995); media, passaging and cryopreservation were as described by these authors except
that lots of heat-inactivated (56° C for 30 min) fetal bovine serum
(FBS), used at 16% concentration, were pretested for ability to
support growth of established lymphoblastoid cell lines at concentrations of approximately 200 000 cells per ml. Cryopreservation
(freezing at approx. 1° C per min and rapid thawing) was at concentrations of about 500 000 cells per ml (viability: 90–95%) in
RPMI 1640 with 10% dimethylsulfoxide (DMSO) and 16% FBS.
For determinations of sensitivities to 4-nitroquinoline-1-oxide
(4NQO), exponentially or near exponentially growing cultures
(> 95% viable cells by trypan dye exclusion) were dispersed by repeated pipeting, counted with a hemocytometer, washed twice in
RPMI 1640 medium without serum and suspended in that medium
at concentrations of 250 000 cells per ml. One ml of the cell suspension was placed in each well of 12-well plates (Corning Costar,
Cambridge, Mass.). An additional 1 ml of this medium was added
with the appropriate concentration of 4NQO. Stock concentrations
of 4NQO (Sigma, St. Louis, Mo.) were at 3 mg/ml in DMSO at
–20° C. Control wells contained DMSO solvent. The wells were
prepared in triplicates for each dose or dose time under consideration. Incubations of the 4NQO-treated cultures were at 37° C in a
humidified incubator in an atmosphere of 5% CO2 in air for 1 h, after which time the cells were resuspended in medium with 16%
FBS and allowed to grow for 24 and 48 h before the cells in each
well were counted (hemocytometer, without staining). To determine the proportions of live versus dead cells and the extent of cell
debris, flow cytometric methodologies were employed using dual
staining by Hoechst 33342 and propidium iodide (Stohr and VogtSchaden 1980). After exposure to 4NQO (protocol as described for
Fig. 1), the cultured B cell lines were harvested by vigorous pipeting, aliquots of 100 000 cells were suspended in tubes with 10 µM
Hoechst 33342 dye and 5 µg per ml propidium iodide and incubated at 37° C in the dark. After a 30-min incubation 40 000 cells
were analyzed using a Coulter Epics Elite Flow Cytometer (Coulter, Hialeah, Fla.) with dual 20 mW UV and 15 mW 488 nm argon
laser excitation. Data were analyzed using MultiPlus software
(Phoenix Flow Systems, San Diego, Calif.) by framing three pop-
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Table 1 Lymphoblastoid cell lines from Werner syndrome (WS)
pedigrees (+/+ wild type, +/– heterozygous carrier, –/– homozygous mutant, WS patient)
LGS family (USA): stop codon mutation in exon 9 at nt 1336
(CGA Arg→TGA Stp) (Oshima et al. 1996)
Registry codea
Genotype
LGS90610
LGS90660
LGS90690
–/–
+/–
+/+
AUS family (Austria): 1-bp deletion (A) in exon 9 at nt 1395 and
exon 30 deletion at nt 3691-3803 due to splice acceptor site mutation (gt→ga) (Oshima et al. 1996)
Registry code
Genotype
AUS40025
AUS40010
–/– (compound heterozygote)
+/– (1-bp deletion)
CWW family (USA):
2-bp deletion in exon 25 at nt 3265-3266
(GA)
4-bp deletion in exon 25 at nt 3259-3262
(CAAA)
Registry code
Genotype
CWW91001
CWW91002
CWW91003
–/– (compound heterozygote)
+/– (2-bp deletion)
+/– (4-bp deletion)
ulations of cells: Hoechst- and propidium iodide-positive cells
with G1 through G2 DNA contents (dead cells), Hoechst- and propidium iodide-positive cells with less than G1 DNA contents (cell
debris), and Hoechst-positive, but propidium iodide-negative cells
(live cells). Apoptosis was measured in cells 24 h after exposure to
4NQO by the detection of a subdiploid DNA content peak (Gong
et al. 1994) and by SYTO 11/Hoechst 33342/propidium iodide
flow cytometry (Poot et al. 1997). For the former, cells were harvested, fixed in ice-cold ethanol overnight, then extracted with
phosphate-citrate buffer and stained with 10 µg DAPI per ml, as
described (Gong et al. 1994). Apoptotic cells were identified by
their appearance as a subdiploid DNA content peak (Gong et al.
1994). In parallel, 4NQO-treated cells were stained for 30 min at
37° C in the dark with 100 nM of the thiazole orange derivative
dye SYTO 11, 5 µg/ml propidium iodide and 10 µM Hoechst
ZM family (Japan): exon 26 deletion at nt 3370-3464 due to splice
donor site mutation (ag→ac) (Yu et al. 1996)
Registry code
Genotype
ZM90630
ZM90633
–/–
+/+
SY family (Japan): stop codon mutation in exon 30 at nt 3724
(CAG Gln→TAG Stp) (Yu et al. 1996)
Registry code
Genotype
SY90575
SY90576
SY90579
–/–
+/–
+/+
SYR family (Syria): 4-bp deletion in exon 32 at nt 3919-3922
(ACAG) (Yu et al. 1996, 1997; Oshima et al. 1996)
Registry code
Genotype
SYR10001
SYR10003
SYR10006
SYR10007
SYR10008
SYR10009
SYR10010
SYR10011
SYR10012
+/–
+/+
–/–
–/–
–/–
+/–
+/+
–/–
+/–
a
Registry codes are those of the International Registry of Werner
Syndrome, University of Washington (G. M. M. and J. O.)
Fig. 1 Lymphoblastoid cell lines were exposed for 1 h to different
concentrations of 4-nitroquinoline-1-oxide (4NQO) in serum-free
medium and incubated in serum-containing culture medium for 24 h
(A) or 48 h (B). The data are expressed as fractions of the cell
counts observed without 4NQO and plotted as the means and standard errors of the means of three independent experiments, two of
which were done by an investigator “blinded” to the diagnoses.
Cell lines were from members of the six Werner syndrome pedigrees in Table 1. solid circles represent data from cultures established from homozygous normal subjects; solid squares are from
homozygous mutant or compound heterozygous mutant Werner
syndrome patients; open circles are from heterozygotes. Each
point represents the mean of 24 cell counts for Werner subjects (n
= 9, with 3 duplicate and 6 triplicate counts), 10 cell counts for
wild-type controls (n = 5, including cultures from subject SYR
10010 used for two experiments, with 3 duplicate and 3 triplicate
counts) and 24 cell counts for heterozygotes (n = 8, with 2 duplicate and 6 triplicate counts)
123
33324 dye. Flow cytometry was performed as described above, detecting live apoptotic cells by their exclusion of propidium iodide
(red fluorescence), cell cycle-specific staining with Hoechst 33324
(blue fluorescence) and reduced staining by SYTO 11 (green fluorescence). This method identifies the same apoptotic population of
cells as is detected by methods that measure reduced mitochondrial
function (Poot et al. 1997).
Results and discussion
Figure 1 summarizes the results of three independent experiments in which the numbers of cells from lymphoblastoid cell lines established from members of the six independent pedigrees listed in Table 1 were determined after exposure to 1 h treatments of various concentrations of
4NQO. Both for the cultures assayed at 24 h (Fig. 1 A) and
48 h (Fig. 1 B) after treatment, lines from heterozygous individuals can clearly be differentiated from the more sensitive homozygous mutants and the more resistant wildtype cells. There are no overlaps of the standard errors of
the means.
Previous studies have indicated altered cell cycle kinetics in WS fibroblasts (Takeuchi et al. 1982) and in WS
Fig. 2 A–C Increase in percentage of dead cells (A) and debris
(B) was quantitated by flow cytometry 72 h after exposure to
4NQO. Cultures of cells from
patients with mutant (–/–) and
wild-type genotypes (+/+) from
three families with WS (TUR,
SY and SYR) were treated with
0.4 or 0.8 µg/ml 4NQO, cultured for 72 h in the absence of
drug, harvested, stained with
Hoechst 33342 dye and propidium iodide and analyzed by
flow cytometry as described in
Materials and methods. Results
from the three families examined are shown as means + SEM.
Statistical significance between
genotypes by paired t test are:
P < 0.04 (dead cells, 0.4 µg/ml
4NQO), P < 0.03 (dead cells,
0.8 µg/ml 4NQO), P < 0.01
(debris, 0.4 µg/ml 4NQO), P <
0.05 (debris, 0.8 µg/ml 4NQO).
As shown in C, cells from patients of one family (SYR) were
examined for the presence of
apoptosis 24 h after exposure to
4NQO, as detected by either the
presence of a subdiploid DNA
content peak (Gong et al. 1994)
or by SYTO 11/Hoechst 33342/
propidium iodide flow cytometry (Poot et al. 1997). Both
methods showed that homozygous mutant cells (–/–) exhibit
higher levels of apoptotic cells
after treatment with 0.4 or 0.8
µg/ml 4NQO
lymphoblastoid cell lines (Poot et al. 1992). We therefore
wished to obtain evidence that the differential declines in
cell counts seen in Fig. 1 were related in part to cell toxicity and cell death, and were not merely the result of inhibition of cell proliferation. Flow cytometric assays were
carried out to assess the extent of cell death and cell debris. For these experiments, only wild-type and homozygous mutant cultures were employed. The results are
shown in Fig. 2. Cell lines from WS subjects, when challenged with 4NQO, undergo more cell death and develop
more cell debris. To investigate further the mode of cell
death, we examined apoptosis in wild-type and homozygous mutant cells from members of one family (SYR).
Similar results were obtained by two cytometric methods
(Fig. 2 C), indicating the presence of higher levels of
apoptosis in mutant cells 24 h after exposure to 4NQO.
Taken together, the data in Fig. 2 suggest that the observed
differential effects of 4NQO on cell survival (Fig. 1) are
due to preferential induction of apoptosis, cell death and
cell degeneration in WRN mutant cells.
The first evidence of hypersensitivity of WS somatic
cells to 4NQO was shown by Gebhart et al. (1988), who
used cultures of mitogen-stimulated peripheral blood T
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lymphocytes. Their cytogenetic studies revealed increased
numbers of chromatid breaks, isochromatid breaks and
chromosomal interchanges in WS cells exposed to 4NQO.
There was some degree of hypersensitivity to diepoxybutane but no hypersensitivity to bleomycin. Previous research with skin fibroblast-like WS cells had revealed
normal responses to UV and X-irradiation (Fujiwara et al.
1977). Given the recent positional cloning of the WRN
gene, one is now in a position to better define various phenotypes, particularly as one can now diagnose heterozygous carriers. To our knowledge, this is the first demonstration of a phenotype in such carriers.
The DNA-damaging, mutagenic and carcinogenic effects of 4NQO require reduction to the 4-hydroxyaminoquinoline-1-oxide (4HAQO) by pyridine dinucleotide
oxido-reductases, including DT-diaphorase (Nagao and
Sugimura 1976; Miller 1991). Subsequent activation of
4HAQO requires esterification by seryl- and prolyl-tRNA
synthetases. These esters alkylate guanine bases (and, to a
lesser extent, adenine bases) in nucleic acids. Given the
putative helicase function of the WRN protein, recent evidence that the degree of reactivity is a function of the
secondary structure of DNA is of interest. Rudolfo et al.
(1994) inserted a poly (dC-dG) sequence in pBR322. There
was reaction of guanine residues with an active 4NQO derivative only when the test sequence was in the B conformation (relaxed conformation). The test sequence adopted
a Z conformation when the DNA was naturally supercoiled, in which state hyperactivity was observed at the BZ junctions. 4NQO is also a potent redox-cycling agent; it
is 10 times more potent than paraquat in its induction of
the oxidative stress-responsive soxRS regulon of Escherichia coli (Nunoshiba and Demple 1993). There are thus
several interesting lines of experimentation to determine
the mechanisms of hypersensitivity of WS cells to 4NQO.
Such research would have significance that goes beyond
the pathogenesis of this rare recessive disorder. First, as
mentioned, the prevalence of heterozygous carriers is sufficient to warrant concern for exposures of carriers to environmental agents with overlapping genotoxic specificities. We need more research on the phenotypes of heterozygous carriers, particularly a follow-up of the work of
Goto et al. (1981), suggestive of an enhanced cancer risk
of WS siblings. Second, gene action at the WRN locus,
which is likely to be related to DNA metabolism, may be
relevant to the differential sensitivities of the general population to common age-related disorders such as certain
forms of cancer (Goto et al. 1981, 1996) and myocardial
infarction (Ye et al. 1997). It is a challenge for the future
to discover why mutations at the WRN locus lead to phenotypes that can be so clearly differentiated from those resulting from mutations at other helicase loci (Ellis et al.
1995; Lombard and Guarente 1996; Epstein and Motulsky
1996).
Acknowledgements We thank Netta Smith and Sue Fredell for
assistance with establishment of B lymphocyte cell lines. The research was supported by NIH grants to G.M.M. (R37 AG 08303
and P01 AG01751) and to P.S.R. (P30 AG13280, Nathan Shock
Center of Excellence for Basic Research on the Biology of Aging).
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