* AGENE Research Institute, Kamakura, Kanagawa 247, Japan; Faculty of Pharmaceutical Sciences, Tohoku University,
Aoba-ku, Sendai 980, Japan; § Tokyo Metropolitan Otsuka Hospital, Toshima-ku, Tokyo 170, Japan
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Abstract |
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We prepared several monoclonal antibodies (mAbs) specific for the NH2- and COOH-terminal regions of the DNA helicase (WRN helicase) responsible for Werner's syndrome known as a premature aging disease. With these antibodies, we detected by immunoblot analysis the endogenous WRN helicase of a relative mass of 180 kD in several lines of cultured cells, but not in patient cells with a defined mutation. Immunocytochemical staining of proliferating fibroblasts and tumor cells showed that the major part of WRN helicase is in the nucleoplasm and not in the nucleolus. Similar experiments with a rat mAb specific to the mouse homologue of human WRN helicase yielded an identical conclusion. Although this nucleoplasmic staining was evident in cells in interphase, the condensed chromatin structure in metaphase was not stained by the same mAbs, suggesting that WRN helicases exist perhaps in a soluble form or bound to the unfolded chromatin structure. From quantitative immunoblot analysis, higher levels of WRN helicase were observed in all transformed cells and tumor cells examined than those of normal cells. The expression of WRN helicase was enhanced consistently in fibroblasts and B-lymphoblastoid cells by transformation with SV-40 and Epstein-Barr virus, respectively, suggesting that rapidly proliferating cells require a high copy numbers of WRN helicase.
Key words: Werner's syndrome; RecQ DNA helicase; genetic instability; aging; nucleoplasmic localization ![]() |
Introduction |
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WERNER'S syndrome (WS)1 is an autosomal recessive disorder causing symptoms of premature aging accompanied by rare cancers. Patients with
WS have a short stature, juvenile cataracts, atrophy of the
skin, graying and loss of hair, diabetes mellitus, arteriosclerosis, osteoporosis, and neoplasia (Werner, 1904; Epstein et al., 1966
; Martin, 1978
; Goto et al., 1996
). The
early appearance of these symptoms generally associated
with normal aging suggests that WS may represent an accelerated aging, with a 2-2.5-fold faster rate than normal
aging. The life span of WS patients is 46 ± 11.6 yr due
mainly to death caused by malignant tumor or cardiovascular infarctions (Goto, 1997
). In vitro studies on the
growth characteristics of fibroblast cells also suggest that
WS may be related to normal aging: the life span of WS
fibroblasts as expressed by population doubling levels
(PDL) is much shorter than that of normal fibroblasts (Salk et al., 1985
). We have already shown that the hypermutator phenotype, such as genetic instability, also occurs
frequently in WS fibroblasts and lymphoblastoid B cells
(B cells) transformed by the Epstein-Barr virus (EBV;
Tahara et al., 1997
).
The gene responsible for the WS (WRN) has been identified by positional cloning from the 8p11-12 region, and
encodes a DNA helicase belonging to a RecQ DNA helicase family (Yu et al., 1996). Defective DNA metabolism
is involved in a complex process of premature aging in WS
patients, because DNA helicases are enzymes that unwind the energetically stable double-stranded structure of DNA
to provide the single-stranded template for important cellular processes like replication, recombination and repair
(Lohman, 1993
). Using an expression system with insect
cells and baculovirus, we and others previously demonstrated that the WRN helicase gene indeed encodes a functional DNA helicase that shows DNA-dependent ATPase and DNA unwinding activities (Gray et al., 1997
; Suzuki et
al., 1997
). We also demonstrated that WRN helicase proteins tagged either with NH2-terminal FLAG peptide or
enhanced green fluorescent protein (EGFP) localize exclusively in the nuclei of HeLa cells, consistent with the
role of DNA helicase in nuclei (Matsumoto et al., 1997a
;
Suzuki et al., 1997
).
Mutations occurring in WS patients have been extensively investigated by us and others with >100 patients. To
date, ~20 different types of mutations have been identified that are distributed in the entire coding regions of the
WRN and exist in individual patients as either homozygous or compound heterozygous mutations (Matsumoto et al., 1997b; Oshima et al., 1997). More recently, we found
that most of these mutations generate truncated helicase
molecules lacking nuclear localization signals (NLS) in the
COOH-proximal region, rendering all WRN products unable to be transported to the nucleus where the DNA helicase is supposed to function (Matsumoto et al., 1997a
;
Matsumoto et al., 1998a
). Importantly, this finding clearly
explains why WS patients show a set of similar clinical phenotypes no matter what type of mutation they carry.
Also, we found that both fibroblasts and EBV-transformed B cell lines from patients with homozygous mutation-4 or mutation-6, two of the most common mutation
types in Japanese WS patients, have much less WRN
mRNA than normal cells (Yamabe et al., 1997). This reduction in the WRN mRNA level suggests an augmented
specific degradation of the WRN mRNA in patient cells,
as was shown in other cases in which the nonsense codons
affect RNA metabolism in vertebrate cells (Maquat, 1995
).
As regards the transcriptional regulation of WRN gene,
we have recently characterized the promotor activity and
found that the WRN gene expression is directed mainly by
the SP1 transcriptional control system, like that of other housekeeping genes, but is further modulated by transcriptional factors, including Rb and p53 (Yamabe et al., 1998
).
In this study, we established several mouse mAbs specific to WRN helicase, and their epitope regions were determined. With these defined antibodies, we investigated the subnuclear distribution of endogenous WRN helicases by immunocytochemical staining. In addition, the levels of endogenous WRN helicase expression were compared for the first time at the protein level for normal cells, transformed cells and for tumor cells. Strikingly, the expression level was highest for immortal EBV-transformed B cells, followed by high levels for mortal EBV-transformed B cells and a low level for untransformed B cells in the peripheral blood. Cellular WRN helicase levels determined by mAbs for other tumor cell lines are discussed in relation to the potential biological function of WRN helicase whose defects apparently result in chromosomal instability in WS patient cells.
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Materials and Methods |
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Cell Lines
Skin fibroblasts were obtained from human biopsy samples and cultured
by the method of Martin (1978); B cells were obtained by transforming
peripheral blood leukocytes with EBV (Miller, 1990
). In brief, peripheral
blood leukocytes from normal individuals and WS patients were purified
using a Lymphocyte Separation Medium and were transformed by EBV
propagated in marmoset cell line B95-8 in the presence of 0.2 µg/ml Cyclosporin (Sandoz). Established cell strains were cultured in 25-cm2 bottles (Corning) using RPM11640 medium (Nissui Pharmaceutical Co. Ltd.)
supplemented with 10% fetal calf serum (GIBCO BRL). The WI38 cell
line originally from human embryonic lung fibroblasts and the WI38 line
transformed by SV-40 were donated by Dr. Ide (Hiroshima University).
Other cell lines were purchased from the American Tissue Culture Collection. All cell lines were maintained in RPMI 1640, McCoy's 5a medium or
DME supplemented with 10% heat-inactivated FCS. The cell lines were
cultured at 37°C in a humidified CO2 incubator.
Separation of Peripheral B-Lymphocytes
We used the panning method (Lewis and Kamin, 1980) to separate B-lymphocytes. In brief, leukocytes were collected from 20 ml peripheral blood
by centrifuging with a lymphocyte separation medium (Organotechnika)
following the instructions of the manufacturer and then suspended in PBS
containing 5% FCS, and were added to a polystyrene culture flask whose
surface had been coated with affinity-purified rabbit anti-human immunoglobulin antibodies (Organotechnika). Leukocytes were kept for 1 h at
20°C. After removing nonadherent cells by decantation and washing five
times with PBS, the adherent B-lymphocytes were recovered from the surface of the culture bottle by leaving the cells 15 min at 20°C in PBS containing 4 mg/ml Lidocain-HCl and by subsequent pipetting. The peripheral blood B cells thus obtained were washed by PBS and were used for
Western blot analysis.
Immunization and Preparation of Hybridomas
To prepare an immunogen, the whole WRN helicase molecule that contained NH2-terminal hexahistidine was produced in insect Sf21 cells with
baculovirus expression systems as described by Suzuki et al. (1997). The
expressed WRN helicase was purified to homogeneity by affinity column
chromatography using Ni-chelating NTA agarose gel (Qiagen Inc.). In addition, a recombinant partial fragment containing NH2-terminal hexahistidine and the COOH-terminal half of WRN helicase consisting of amino
acid residues 877-1,432 was produced in E. coli, and was used as an immunogen after purifying by affinity chromatography with NTA resin. Rat
mAbs specific to the mouse homologue of human WRN helicase were
made using the purified protein, containing NH2-terminal glutathione-
S-transferase, and the COOH-terminal polypeptide fragment consisting
of amino acid residues 273-513 of mouse WRN helicase, which were made
in E. coli. BALB/c mice and Wistar rats were, respectively, immunized with these purified proteins in a complete Freund's adjuvant. Hybridoma
clones that produced the antigen-specific antibodies were screened by
ELISA and Western blot analysis.
Determination of the Epitope Regions in the WRN Helicase Molecule Recognized by mAbs
Various NH2- or COOH-terminal fragments of the WRN helicase protein
that were NH2-terminally tagged with EGFP were produced in mouse
melanoma cells B16F10 by transfecting a series of recombinant pEGFP-C3 expression plasmids (Clontech Laboratories Inc.) that contained the
CMV promoter and the downstream DNA sequences coding for EGFP
and fragments of WRN helicase. For example, the C49 and C59 polypeptides that contain NH2-terminal EGFP and COOH-terminal fragments of
WRN helicase consisting of 49 aa and 59 aa, respectively, were generated
by placing the cognate DNA fragments coding for these polypeptides in
pEGFP-C3 plasmid downstream of the CMV promoter-EGFP sequence,
and were transiently expressed in B16F10 cells by transfecting the constructed recombinant plasmid DNA (Matsumoto et al., 1997a). The N368
polypeptide containing the NH2-terminal 368 aa was similarly produced in
B16F10 cells. Expression plasmids coding for other EGFP-conjugated
proteins, N1304, N1162, N1046, C138, C128, C79, C69, and C19, were
similarly constructed and were expressed in B16F10 cells. Cells expressing these polypeptides were fixed as described above in the immunocytochemistry section and were stained with the mAbs conjugated with fluorescent reagent Texas red. To define the epitope sites more precisely for
mAbs to recognize the NH2-terminal region of WRN helicase, a recombinant partial fragment (C1201) containing NH2-terminal hexahistidine, but
with no NH2-terminal 231 aa, was synthesized and was used for Western blot analysis after purification by Ni-NTA column chromatography.
Immunoblot Analysis
Cell lysates were prepared as described by Tada et al. (1996) from subconfluent cultures of cells. Approximately 107 cells were washed with ice-cold
PBS, pelleted and disrupted in RIPA buffer containing 10 mM Tris-HCl
buffer (pH 7.4), 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM ethylene diamine tetra acetate and a cocktail of protease
inhibitors (PharMingen), and a portion of cell lysate containing 20 µg protein (derived from ~1.5 × 105 fibroblast cells and 2.2 × 105 lymphoblastoid B cells) was electrophoresed on 7% SDS-polyacrylamide gels. A
prestained SDS-PAGE standard protein marker (Bio-Rad Laboratories Inc.) was used to calibrate the molecular mass; it contained myosin (213 kD),
-galactosidase (119 kD), BSA (83 kD), and ovalbumin (47 kD).
Proteins resolved in gel were electrophoretically transferred to a polyvinylidene difluoride membrane, Immobilon (Millipore). The transferred
membrane was treated with TBS (20 mM Tris-HCl, 150 mM NaCl, pH
7.5) containing 5% (wt/vol) skim milk for 1 h. The membrane was then incubated overnight at 4°C with diluted antibodies to determine the specificity and affinity to WRN helicase of the antibodies. The membrane was
washed and then was incubated with anti-mouse IgG-conjugated HRP
(Dako). After washing again, the membrane was developed using ECLTM
(Amersham Life Science, UK).
Immunocytochemistry by Indirect Immunofluorescence
Cells were grown to ~80% confluency in 100-mm tissue culture dishes (Corning) and were stuck onto a silane-coated slide by cytospin. The spread cells were immediately fixed with 3.7% formaldehyde in PBS for 10 min and washed and permeabilized with PBS containing 0.05% Tween 20 (washing buffer). They were then treated with 3% (wt/vol) skim milk in PBS at room temperature for 2 h, treated with 0.1% Triton X-100 in PBS, and incubated with mouse anti-WRN helicase mAbs as the primary antibodies overnight at 4°C. The cells were washed again with the washing buffer three times for a total of 30 min, and they were further incubated with biotinylated goat anti-mouse IgG1 (Southern Biotechnology Associates Inc.) for 1 h at room temperature. The cells were washed again with the washing buffer and incubated with 5 µg/ml streptavidin FITC (PharMingen) for 30 min at room temperature.
Double immunocytostaining was made for WRN helicase and the nucleolus protein using mouse mAb 8H3 and the goat polyclonal antibody B23 (Santa Cruz) specific to nucleolar nucleophosmin/B23 protein as primary antibodies. These antibodies were mixed and incubated with the fixed HeLa cells. As secondary antibodies, the biotinylated donkey anti- mouse IgG and the Cy3 conjugated donkey anti-goat IgG (both from Chemicon International) were used to detect the primary antibodies that bound to WRN helicase and the nucleolus protein. The Cy3-conjugated IgG emitted red fluorescence, while the biotinylated anti-mouse IgG was stained green by streptavidin FITC by the same conditions described above. The nuclei were counterstained blue with DAPI. Fluorescent images were visualized with a Bionanoscope (Nikon Engineering) fitted with a 100× Nikon PlanApo oil immersion objective and two double-pass filter sets for fluorescein/DAPI and Texas red. An alternative procedure using acetone-methanol (1:1 vol/vol) was also carried out, which resulted in similar staining profiles (data not shown).
Determination of Subnuclear Distribution of WRN Helicase by Expression of the Full-Size cDNA in HeLa Cells
An expression plasmid was constructed with the full-size WRN cDNA
and pEGFP-C3 plasmid (Clontech). The cloned full-size cDNA was
placed downstream of the EGFP sequence in the pEGFP-C3 DNA, according to the manufacturer's instruction. It was transfected to HeLa cells
and the WRN helicase NH2-terminally tagged with EGFP was generated
as described before (Matsumoto et al., 1997a). HeLa cells were seeded
onto a poly-L-lysine-coated cover glass (Iwaki Co.), cultured overnight
and then transfected with 5 µg of expression plasmid DNA mixed with 10 µg
lipofectin (GIBCO BRL). 24 h after transfection at 37°C, the cells were
fixed with 4% paraformaldehyde. A cover glass was mounted with 0.2 µg/ml
DAPI in antifade solution (Wako). The EGFP fusion proteins and nuclei were visualized by fluorescence microscopy with a Nikon Optiphot-2 microscope fitted with a 60× Nikon PlanApo oil immersion objective and a
triple-pass filter set for fluorescein/DAPI and Texas red. Fluorescent images were collected using a high performance CCD camera and were processed by MacProbe (Perceptive Scientific Instruments).
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Results |
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Preparation of mAbs Specific for WRN Helicase and Epitope Mapping
After screening multiple independent clones of hybridoma
cells, we found five hybridoma cell lines generating the
isotypes of mouse IgG1, referred to hereafter as 3D12,
4H12, 4D9, 4F8, and 8H3, that bind efficiently in immunoblot analysis to whole molecules of purified WRN helicase
(Fig. 1). Their epitopes were determined by measuring the
immunoreactivity of mAbs to parts of the WRN helicase
generated by expression of the fragments of WRN cDNA
in B16F10 mouse melanoma cells (Table I). The B16F10 cells express a very low level of endogenous murine WRN
helicase protein (our unpublished data), and therefore do
not disturb the detection of binding between mAbs and
the recombinant derivatives of human WRN helicase expressed in the cells. The derivatives of human WRN helicase contained NH2-terminal EGFP and various lengths of
fragments of WRN helicase (aa residues 1-368, 1-1,046, 1-1,162, 1-1,304, 1,294-1,432, 1,304-1,432, 1,353-1,432,
1,363-1,432, 1,373-1,432, 1,383-1,432, and 1,413-1,432). A
convenient color reaction was used to assess the epitopes
of each mAbs; the location of expressed fusion proteins
were shown by green fluorescence, and positive immunoreactions between fusion proteins and mAbs were shown
by a red color due to Texas red-conjugated avidin that binds to biotinylated anti-mouse IgG (Fig. 2). mAb 4H12
was found to react with COOH-terminal polypeptide C59
consisting of 59 aa residues but not to a shorter COOH-terminal polypeptide C49 consisting of 49 aa residues, indicating that the epitope of 4H12 is in the 10 aa residues
that C49 lacks (Fig. 2 A). This 10-aa region contains the
NLS required for WRN helicase to migrate from the cytoplasm to the nucleus (Matsumoto et al., 1997b
). The mAbs
4D9, 4F8, and 8H3 could bind to the N368 polypeptide
containing the NH2-terminal 368 aa residues of WRN helicase (Fig. 2 B shows the data obtained with mAb 8H3).
The epitopes of 4D9, 4F8, and 8H3 were further narrowed
by immunoblot analysis to a NH2-proximal region between amino acid residues 232 and 368 after examining
their binding activities to a mutant WRN helicase (C1201)
that lacked NH2-terminal aa residues 1-231 (Fig. 2 C).
This region is downstream of the 3'-5' exonuclease region
(60-231 aa residues) predicted by Mushegian et al. (1997)
from their homology search studies. The mAb 3D12 bound to recombinant WRN helicase proteins encompassing the helicase domain and the COOH terminus (877-
1,432 aa residues), suggests that its epitope is on the
COOH-terminal side. Table I summarizes these results.
Two mAb, 8H3 and 4H12, that recognize distinct epitopes
on WRN helicase at NH2-terminal 232-368 aa and
COOH-terminal 1,373-1,432 aa, respectively, were mainly
used for the following studies.
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Detection of WRN Helicase Molecules in Fibroblasts and EBV-transformed B-Lymphoblastoid Cells from Normal Individuals
To detect endogenous WRN helicase in fibroblast cells from normals and WS patients, we first carried out an immunoblot analysis using mAb 4H12 that has the epitope in the COOH-terminal region of the WRN helicase. Fig. 3 A shows that mAb 4H12 detected WRN helicase with a molecular mass of 180 kD in the cell lysates prepared from normal individuals (lanes 3 and 4). As expected, no specific band to WRN helicase was seen in the patient cell lysates from homozygous gene mutation type 4/4 that yields a truncated protein (1-1,060 aa) lacking the epitope of 4H12 (lanes 1 and 2). To find out if this immunological detection extends to more easily obtainable EBV-transformed B cells, we used immunoblot analysis for B cells from two WS patients and two normals using mAb 8H3 that has an epitope in the NH2-terminal region (Fig. 3 B). Like fibroblast cells, the cell lysate of B cells from normal individuals showed a clear band of 180 kD WRN helicase (lanes 3 and 4). In contrast to a clear visualization of WRN helicase in the cell lysate prepared from the cells of normal individuals, we found no sign of immunoreactive WRN helicase-specific polypeptide in the cell lysates from WS patients carrying a homozygous mutation 4 (lanes 1 and 2). The amount of WRN helicase protein in 20 µg lysate protein of B cells was greater than that of fibroblast cells, as estimated from the intensities of the stained bands in immunoblot analysis.
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Upregulation of the Expression of WRN Helicase by Transformation and Immortalization
The expression levels of the WRN gene product in fibroblasts derived from normal individuals were about one-tenth of those of EBV-transformed B-lymphoblastoid cells (Fig. 3). To gain more information about the levels of WRN helicase protein in various cells, we compared the levels of WRN helicase protein for the WI38 human diploid cell line derived from embryonic lung and the same WI38 cells transformed with SV-40. Comparative immunoblot analysis showed that the SV-40-transformed WI38 cells (WI38/SV) contained a higher level (~10-fold) of WRN helicase protein than the original WI38 cells (Fig. 4 A, lanes 1 and 2). The level in WI38/SV cells was comparable to or slightly greater than that in HeLa cells (lane 3).
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The transformation of B cells by EBV can only extend
the life span of most B cells up to a maximum 160 PDLs
and does not necessarily make the cells immortal, as opposed to an often misunderstood concept that EBV infection makes B cells immortal (Tahara et al., 1997). However, a few populations of immortal B cells occur after a
long period of culture of transformed B cells, similar to fibroblast cells transformed by SV-40. In our laboratory,
three such immortalized B-lymphoblastoid cell lines were
established from B cells transformed by EBV originally
from normal individuals after a long period of culture, and
the cells showed strong telomerase and infinitely proliferating activities (Kataoka et al., 1997). About twofold higher levels of WRN helicase were observed with these
immortalized B cells (Fig. 4 B, lanes 4 and 6) than with
their parental cells, which were mortal (lanes 3 and 5). In
a separate comparative study, we examined the level of
WRN helicase in untransformed B cells purified from peripheral blood cells, but no WRN helicase protein was detected (Fig. 4 B, lanes 1 and 2). Here, the order of WRN helicase expression was (a) immortal EBV-transformed
B cells with telomerase activity, (b) mortal EBV-transformed B cells with no or very low telomerase activity, (c)
untransformed B cells in peripheral blood. The data obtained with B cells predict that tumor cells may have
higher levels of WRN helicase, as they are transformed
and immortalized from original normal progenitor cells.
WRN Helicases in Tumor Cells
The expression levels of WRN helicase were compared in several human tumor cell lines by comparative immunoblot analysis with mAb 4H12 that recognizes the COOH-terminal epitope. All human tumor cell lines examined showed a level equivalent to that in HeLa cells or higher levels (Fig. 4 C). These cell lines were HT1080 (fibrosarcoma), PA1 (ovarian teratocarcinoma), Tera2 (embryonic carcinoma), T24 (bladder carcinoma), K562 (chronic myelogenous leukemia), Molt4 (acute lymphoblastic leukemia), and A172 (glioblastoma) cells, and are shown in lanes 2, 3, 4, 5, 6, 7 and 8, respectively (Fig. 4 C). The highest expression was found in K562 cells that showed much higher activity than that of HeLa cells (lane 6) followed by PA1, Tera2, and Molt4 cells (lanes 3, 4, and 7). The molecular size of WRN helicase was unaltered and remained at 180 kD for all tumor cells examined.
Immunocytochemistry Showing the Location of WRN Helicase in the Nucleoplasm
We used mAbs to determine the subnuclear location of
WRN helicase. Previously, we showed that WRN helicases
expressed in HeLa cells by transfected expression plasmids migrate exclusively to the nucleus (Matsumoto et al.,
1997a; Suzuki et al., 1997
). This nuclear migration was
found to be dependent on the NLS encoded in the COOH-terminal region of WRN helicase (Matsumoto et al., 1998a
). However, where in the nucleus WRN helicases reside, and whether the location changes upon cell cycling,
remained unclear. To define the subnuclear location, we
immunocytochemically stained the endogenous WRN helicase in skin fibroblasts from a normal individual (Fig. 5 I,
A and B), skin fibroblasts from a WS patient (E and F),
Tera2 cells (I and J), and WI38/SV cells (O and P). All the
results showed that WRN helicase was mainly located in the nucleoplasm. In these cells the nucleolus regions were
left unstained and were represented by black holes (Fig. 5
I, J and P). A higher magnification of the immunostaining
of Tera2 cells by mAb 4F8 showed that the stained WRN
helicase antibody exists as multiple nuclear dots (Fig. 6).
Similar profiles were obtained by other mAbs 3D12, 8H3,
and 4D9. To avoid the possibility that nucleolus proteins
might be leaked by disruption of nucleoli during our immunocytostaining, as a reference, we stained the cells similarly with a different anti-human nucleolus antibody AE3
that has been broadly used to stain specifically the nucleolus. However, this was not the case, as shown in Fig. 5 I (C
and D, G and H, and K and L for normal fibroblast, WS fibroblast and Tera2, respectively) in which the nucleoli of
these cells were stained distinctly by the nucleolus-specific
antibodies. When mouse colon carcinoma cells (colon 26)
were stained with a rat monoclonal antibody specific to
the mouse WRN helicase, again only the nucleoplasm was
stained (Fig. 5 I, Q and R), indicating that the major part
of WRN helicase is in the nucleoplasm and not in the nucleoli for both human and mouse cells.
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To confirm the nucleoplasmic localization of WRN helicase, we made a double immunostaining of growing HeLa cells using a mixture of two antibodies, mouse mAb 8H3 specific to WRN helicase and goat polyclonal antibody B23 specific to the nucleolar protein nucleophosmin/B23. The WRN helicase was simultaneously stained green using FITC and the nucleolus protein red using Cy3 (Fig. 7, 2 and 3). Under the conditions described in Materials and Methods, whole HeLa nuclei were stained blue by DAPI (which stains DNA), leaving several nucleolar regions unstained (Fig. 7, 1). The green WRN helicase signals spread in the entire nucleus consistent with a localization of WRN helicase mainly in nucleoplasm (Fig. 7, 2), while the red B23 signals were restricted in the nucleolar regions that were unstained by DAPI (Fig. 7, 3). The overlaid picture (Fig. 7, 4) showed that the nucleolar regions were distinctly stained with red/orange, and the surrounding nucleoplasm was stained with green, indicating nonoverlapping of WRN helicase and the nucleolar nucleophosmin/B23. These double immunocytostaining data confirmed our conclusion that the WRN helicase is mainly in the nucleoplasm of growing HeLa cells and not in the nucleolus.
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Previously, we expressed the full-size WRN cDNA in
HeLa cells to generate the WRN helicase NH2-terminally
tagged with EGFP or FLAG. The expressed FLAG-WRN
helicases localized in the entire nucleus of HeLa cells (Suzuki et al., 1997 [Fig. 2 therein]). In a similar experiment,
the expressed EGFP-WRN helicases also localized in the
nucleus (Matsumoto et al., 1997a
), with a notable exclusion from small regions that presumably are nucleolus
(Fig. 5 I, I). The results of these experiments are consistent
with a conclusion of this paper that the WRN helicases
mainly exist in the nucleoplasm.
To find out if WRN helicase exists in the condensed chromatin at metaphase, we analyzed the distribution of WRN helicase in Tera2 cells stained during their metaphase; however, we were unable to detect WRN helicase on the condensed chromatin (Fig. 5 I, M and N). These data indicate that WRN helicase is distributed in the nucleoplasm, not in the nucleoli or on the condensed chromatin at the metaphase.
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Discussion |
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The gene, its product DNA helicase and various gene mutations responsible for Werner syndrome have been identified. Our previous findings that defective WRN helicase in patient cells are unable to migrate to the nucleus because of a lack of COOH-terminal NLS also explains why patients with WS show similar clinical phenotypes regardless of the type of gene mutation. Despite this fundamental knowledge of the pathogenesis of WS, several important issues concerning the endogenous protein level of WRN helicase in normal cells or cells of various states and its precise subnuclear location remained unknown. This report clarifies some of these issues.
Diagnosis of Werner Syndrome by Immunoblot Analysis
In this study, two types of mAb recognizing the NH2-
or COOH-terminal region of WRN helicase were established. We used these antibodies in Western blot analysis
to make the immunological diagnosis of WS possible by
analyzing the lysates of cells from patients and normal individuals. The WS patients with mutation type-4, the most
frequent WRN mutation in the world, can be diagnosed immunologically by the absence of WRN helicase protein
in EBV-transformed B cell lysates (Fig. 3). Recently, we
developed two types of genetic diagnosis systems, mutant
allele specific amplification and oligonucleotide ligation
assay that use the nucleotide sequence information of mutated genes and PCR (Matsumoto et al., 1998b). However,
because such a genetic diagnosis is made from only known
mutated gene sequences and their specific primer settings,
they can not be used for WS with unknown mutations. To
detect truncated gene products or the absence of gene
products, the immunological method using mAbs with a
refined epitope, as is shown for WS in this report, will lead
the way to the clinical field in combination with a genetic
diagnosis. Most WRN mutations result in the generation of truncated proteins, and therefore, the absence of a 180-kD full-size WRN helicase molecule in EBV-transformed B
cells from patients will indeed indicate WS. Previously, we
reported that the levels of mutant WRN mRNA in WS patient cells are significantly lower (average 16%) than those
of intact mRNA from normal individuals (Yamabe et al.,
1997
), and we anticipated a low level of truncated WRN
gene products in WS patient cells. The results obtained
from patient cell lysates by immunoblot analysis were beyond this anticipation, because the patient cells (with homozygous mutation 4) showed almost no detectable truncated WRN helicase molecules with predicted size of 120 kD.
Our more extended studies showed that cells from WS patients carrying homozygous mutations 6, 7, and 9 also
contained no mutant proteins in the predicted truncated
forms of 40, 130, and 70 kD, respectively (data not shown).
We speculate that the truncated WRN helicase molecules
may be sensitive to cellular proteases, and may be removed from WS patient cells by proteolytic digestion. A
slightly larger molecular mass (180 kD) of endogenous WRN helicase than the predicted 162 kD was observed for
all cells used in this study. Whether this larger molecular
mass is due to phosphorylation and/or other post translational protein modifications should be investigated.
WRN Helicase Gene Expression Is Upregulated in Rapidly Proliferating Cells
Our findings that the copy number of WRN helicase is
markedly augmented in EBV-transformed B cells or SV-40-transformed fibroblasts apparently provide an important foresight into the role of WRN helicase in these cells
that have acquired a rapidly proliferating phenotype. Today, whether the transformation by viruses fortuitously stimulates the transcription and translation of WRN, or
whether the cells that proliferate fast require a high level
of WRN helicase and thus such cells with a large copy
number of WRN helicases are selected, is not clear. The
latter hypothesis is plausible because all the tumor cell
lines that are free from virus infection and proliferate rapidly showed high levels of WRN expression (Fig. 4 C). In
this context, we recently found the transcription of WRN
helicase gene is regulated by several SP1 and a RCE elements, characteristic of a housekeeping gene, but is further modulated by tumor suppressor Rb and p53 proteins:
upregulation by Rb and downregulation by p53 (Yamabe
et al., 1998). The high levels of WRN helicase in tumor
cells observed in this study may be due to the combined effects of the increased Rb and the inactivation of p53 by
mutation. Perhaps, greater numbers of WRN helicase may be needed for some rapidly growing cells to suppress the
genomic instability resulting from the illegitimate recombinations incurred by the continual DNA replications.
Supporting this hypothesis, we recently showed that WRN
gene can suppress the increased homologous and illegitimate recombination of Saccharomyces cerevisiae mutant cells that lack the Sgs1 DNA helicase belonging to the
RecQ helicase family (Yamagata et al., 1998
).
WRN Helicases Are Distributed as Nuclear Dots in the Nucleoplasm and Are Not in the Condensed Chromatin
Our immunocytochemical studies clearly showed that WRN helicases are distributed in the nucleoplasm during the interphase, and are not in the condensed chromatin in the metaphase (Fig. 5 I, M and N). Marciniak et al. (1998), using polyclonal antibodies, showed that WRN helicase localizes in the nucleolus of WI38 cells, whereas the mouse WRN helicase is distributed in the nucleoplasm. Their data on the location of human WRN helicase, therefore, contradict the findings of this study. This discrepancy does not seem to stem from the methods used for fixing cells because we obtained unambiguous nucleoplasmic staining for WI38 and WI38/SV cells after fixation with the acetone-methanol method that Marciniak used. The conditions of the cultured cells, e.g., growing or nongrowing, or the extent of cell confluence, may rather affect the mode of nuclear localization. These important issues remain to be clarified by the specific and refined antibodies characterized in this study. In this context, the fact should be mentioned that the commercially available polyclonal antibody (sc-1956; Santa Cruz) produced a similar result as our mAbs.
Our findings described in this paper are also dissimilar
to previous observations obtained with yeast sgs1 helicase,
a homologue of WRN helicase in S. cerevisiae, in which
sgs1 exists in the nucleolus when the cells are young and
migrates to the nucleoplasm as the cells senesce and the
nucleoli fragment (Sinclair et al., 1997). Rather, the profiles of immunostaining of WRN helicase shown in this report resemble those of p53 and RF-A (replication factor
A) that bind to DNA exclusively in the nucleoplasm (Din et al., 1990
; Wilcock and Lane, 1991
). Further studies are
needed to determine if yeast sgs1 has the same biological
role as human WRN helicases in DNA metabolism. In addition, most importantly, the kind of DNA event(s) where
WRN helicase fails to participate in patient cells remains
to be clarified.
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Footnotes |
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Received for publication 10 July 1998 and in revised form 19 November 1998.
Address correspondence to Yasuhiro Furuichi, AGENE Research Institute, 200 Kajiwara, Kamakura, Kanagawa, 247 Japan. Fax: 81 467 48 6595. E-mail: furuichi{at}agene.co.jp
We thank Ms. Chie Itoh, Kumiko Fujita, Masako Okada, and Mr. Megumu Shio for their technical assistance. We also thank Dr. Aaron J. Shatkin at the CABM for valuable discussions.
This work was supported by the Drug Organization supervised by the Ministry of Health and Welfare of Japan.
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Abbreviations used in this paper |
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aa, amino acids; EBV, Epstein-Barr virus; EGFP, enhanced green fluorescent protein; NLS, nuclear localization signals; PDL, population doubling levels; WS, Werner's syndrome.
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