From the Kimmel Cancer Center, Thomas Jefferson
University, Philadelphia, Pennsylvania 19107, the § Chiron
Corporation, Emeryville, California 94608, and the ¶ Chiron
Corporation and Department of Pharmaceutical Chemistry, University of
California, San Francisco, California 94608
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ABSTRACT |
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3T3 cells null for the type 1 insulin-like growth
factor receptor are refractory to stimulation by a variety of purified
growth factors that are known to be required for the stimulation of
other 3T3 cells. However, these cells, known as R
cells, grow in serum-supplemented medium and also in media conditioned by certain cell lines. We report here the purification of a growth factor that stimulates DNA synthesis (and growth) of R
cells. The growth factor, purified to homogeneity by SDS-polyacrylamide gel electrophoresis, was identified as the granulin/epithelin precursor
by an accurate determination of the masses of endoproteinase Lys-C
peptides using matrix-assisted laser desorption ionization mass
spectrometry, followed by a data base search. The granulin/epithelin precursor is a little known growth factor, secreted by a variety of
epithelial and hemopoietic cells. It is at present the only purified
growth factor that can stimulate the growth of mouse embryo fibroblasts
null for the type 1 insulin-like growth factor receptor.
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INTRODUCTION |
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It has been known for several years that the insulin-like growth
factors (IGF-I and -II)1 play
a central role in the growth of cells in culture (reviewed in Ref. 1).
Most cells in the animal body have type 1 IGF receptors (IGF-IR) and
require the activation of this receptor by its ligands for optimal
growth, both in vivo (2, 3) and in vitro
(reviewed in Ref. 4). The requirement for IGF-I is especially evident with mouse and human fibroblasts, where it combines with other growth
factors (for instance, platelet-derived growth factor) in stimulating
growth of cells under defined conditions (serum-free medium). Singly,
these growth factors cannot stimulate the growth of normal fibroblasts,
like 3T3 cells (5). R cells (6, 7) are 3T3 fibroblasts
that originated from mouse embryos with a targeted disruption of the
IGF-IR genes (2, 3). R
cells have been extensively used
in the past years to study the role of the IGF-IR in mitogenesis,
transformation, and apoptosis (see the editorial in Ref. 8).
R
cells do not grow in serum-free medium supplemented
with the growth factors that are known to sustain the growth of other
3T3 cells, with a physiological number of IGF-IRs (1). Singly or in
combination, the following growth factors failed to stimulate the
growth of R
cells: platelet-derived growth factor,
epidermal growth factor, IGF-I and II, insulin, basic and acidic
fibroblast growth factor, TGF
, TGF
, and hepatocyte growth factor
(7, 9, 10). In fact, even R
cells overexpressing either
the epidermal growth factor receptor (11) or the platelet-derived
growth factor
receptor (12) are unresponsive to their respective
growth factors, indicating a central role of the IGF-IR in fibroblast
growth. Reintroduction of a wild type IGF-IR promptly restores the
growth deficits of R
cells (6, 7). However, the fact that
R
cells grow in 10% serum clearly indicates that serum
contains one or more growth factors that bypass the requirement for
IGFs. R
cells also grow in medium conditioned by certain
(but not all) cell lines (9). In previous papers (9, 13), we reported that the conditioned medium of BRL-3A cells stimulated the growth of
R
cells. The partial fractionation of a
growth-stimulating polypeptide derived from BRL-3A cells was reported
by Xu et al. (9). The growth factor has now been identified
as the granulin/epithelin precursor (14-16) by high accuracy peptide
mass mapping with matrix-assisted laser desorption ionization (MALDI)
mass spectrometry (MS), followed by data base searching with a set of
measured peptide masses. Further evidence for its identity was provided
by sequence analysis of a peptide by Edman degradation after
purification by reverse phase chromatography. Given the central role of
the activated IGF-IR in the growth of cells in vivo and
in vitro (see above), the identification of the
granulin/epithelin precursor as a growth factor that bypasses the
requirement for IGFs can be of considerable interest to the many
investigators in the field of IGFs and IGF-binding proteins and of cell
proliferation in general.
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EXPERIMENTAL PROCEDURES |
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Cell Lines and Culture Conditions--
All experiments used as
the test cell line R cells (6, 7), which are 3T3-like
cells derived from mouse embryos with a targeted disruption of the
IGF-IR genes (2, 3). These cells have been repeatedly described and
characterized in previous papers from this laboratory (11). They grow
in 10% fetal bovine serum, but they do not grow at all in serum-free
medium supplemented with purified growth factors. R
cells
were maintained routinely in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum.
Preparation of Conditioned Medium--
Conditioned medium from
BRL-3A cells was prepared as described in detail by Xu et
al. (9). Collected medium was sterilized using a 0.2-µm filter
and stored frozen at 20 °C until needed.
DNA Synthesis--
R cells were seeded at a
density of 5 × 103/cm2 on coverslips in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
and allowed to attach for 24 h. The cultures were then made quiescent for 96 h in serum-free medium prior to the addition of
growth factors or conditioned medium. Tritiated thymidine (0.5 µCi/ml) was added at the same time as growth factors or conditioned medium, and the incubations were continued for 24 h. The cells were then fixed in cold methanol and autoradiographed by standard procedures. The percentage of labeled cells was determined by scoring a
total of 1,000 cells. DNA synthesis stimulating activity was expressed
as the percentage of labeled nuclei.
Chromatography Procedures-- The purification of the growth factor is schematically represented in Fig. 1.
Polyacrylamide Electrophoresis-- Polyacrylamide electrophoresis of proteins in any sample was performed using Precast Ready Gels and Mini-Protean II cell system (Bio-Rad) unless otherwise noted. 4-20% linear gradient Tris-glycine gels were used for the inset of Figs. 2 and for Fig. 3B, whereas 4-15% linear gradient Tris-glycine gels were used for Fig. 3A and the inset of Fig. 4.
Recovery of Protein Resolved under Nonreducing Condition-- 200 µl of fraction 38 (about 3 µg of protein) from the reverse phase chromatography procedure was lyophilized by Speed-Vac concentrator and resolved by polyacrylamide gel electrophoresis under nonreducing condition. One-sixth of the sample was run onto a separate lane for silver stain as reference. Slices above, below, and corresponding to the desired band detected by silver stain (Fig. 3B) were cut out from the gel and were incubated in 1 ml of phosphate-buffered saline at 4 °C for 12 h. The supernatant was dialyzed by Slide-A-Lyzer 10K Dialysis Cassettes (Pierce) twice against saline for 6 h and twice against distilled water for 6 h (18). The product was concentrated by Centricon-10 (Amicon, molecular weight cut-off of 10,000) unit; four-fifths were tested for the ability to stimulate DNA synthesis, and one-fifth was analyzed by SDS-PAGE under reducing conditions (Fig. 4).
Renaturation of Protein Reduced by SDS-PAGE-- Target protein (3 µg) in 200 µl of fraction 38 from reverse phase chromatography procedure was also studied by cutting out the desired band from the gel after resolution by SDS-PAGE. The protein was eluted from the gel band, renatured, and characterized, basically according to the method of Hager and co-workers (19, 20) and Ishii et al. (21).
Preparation of Protein for Peptide Analysis-- To obtain a homogenous protein preparation for peptide analysis, the active factor was purified by SDS-PAGE. The preparation was lyophilized in a vacuum concentrator, and the protein were subjected to reduction by dithiothreitol and cysteine alkylation by 4-vinylpyridine in Laemmli SDS-PAGE sample buffer. The mixture was applied to a single lane of a 14% Novex precast Tris-glycine gel and subjected to electrophoresis under normal conditions. The protein was visualized by Coomassie Blue stain, the major component was excised, and the gel slice was destained in preparation for endoproteinase digestion in situ (22).
Protein Digestion, Peptide Isolation, and Sequence Analysis-- The protein present in the gel slice was subjected to endoproteinase Lys-C (Achromobacter lyticus from Wako) digestion for 16 h. Peptides were extracted from the gel slice in organic solvents, and 10% of the mixture was subjected to analysis by MALDI reflectron time-of-flight (MALDI-TOF) MS. The bulk of the peptide mixture (90%) was subjected to reverse phase capillary chromatography (Applied Biosystems 173A cLC Microblotter system) with deposition of the peptides on a polyvinylidene difluoride membrane after passage through the detector cell. One peak in the chromatogram was subjected to Edman degradation using an Applied Biosystems Procise 494 protein sequencer through direct introduction of the polyvinylidene difluoride membrane and use of "blot" cycles.
Protein Data Base Search with Peptide Mass Data-- Peptide monoisotopic masses were used for protein data base searching using PeptideSearch software2 running on an Apple Power Macintosh 7600/120.
IRS-1 and MAP Kinase Phosphorylation-- Cells were treated as for DNA synthesis (see above), and lysates were prepared from them for IRS-1 and MAP kinase phosphorylation following techniques previously described (10). For IRS-1, 300 µg of protein lysate were immunoprecipitated overnight at 4 °C with anti-IRS-1 and protein A-agarose (Oncogene Science). After resolution on a 4-15% SDS-PAGE (Bio-Rad) and transfer to a nitrocellulose filter, the membrane was probed with anti-phosphotyrosine antibody (Transduction Laboratories). Detection was carried out with ECL (Amersham Pharmacia Biotech). After membrane incubation with stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 7.6) at 50 °C for 30 m, membrane was washed twice with TBS-T buffer (0.1 M Tris, 1.5 M NaCl, 0.5% Triton Y-100) blocked overnight in 5% nonfat milk in TBS-T buffer and probed with anti IRS-1 antibody (Oncogene Science). MAP kinase phosphorylation was determined following the instructions of the antibody's manufacturer (Promega).
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RESULTS |
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Purification of the Growth Factor--
In a previous paper (9), we
showed that R cells could grow in media conditioned by
three different cell lines, BRL-3A, T98G (a human glioblastoma cell
line), and T24H (a 3T3 cell line transformed by an activated
ras). Other cell lines were also tested and found to be
negative, i.e. their conditioned media did not elicit a
response in R
cells. Among the other cell lines tested
were two normal cell lines, WI-38 human diploid fibroblasts and Balb/c
3T3 cells, and a human breast cancer cell line, MCF-7. In general, the
cells whose conditioned media stimulated R
cells were
cell lines capable of growing in serum-free medium, whereas the
negative cell lines required IGF-I (as well as other growth factors)
for growth. We concentrated on BRL-3A conditioned medium, because it
seemed more potent than other conditioned media in stimulating the
growth of R
cells. The partial purification of the
unidentified growth factor has been described by Xu et al.
(9). A diagram of the purification procedure is given in Fig.
1. At every step, the various fractions were used to stimulate R
cells. Although the activity was
originally identified as capable of causing an increase in cell number
in R
cells (9), in later experiments, the usual procedure
was to monitor each fraction for the ability to stimulate DNA
synthesis. This procedure is easier and more sensitive than cell
number, because even when close to 100% of cells enter S phase, the
increase in cell number is only a doubling. However, the purified
growth factor can also stimulate cell growth, in addition to
stimulating DNA synthesis. A pool of fractions from the
phenyl-Sepharose column that was active in stimulating the growth of
R
cells was further fractionated by reverse phase
chromatography (Fig. 2). The highest
stimulating activity was found in fractions 38-40. When these
fractions were run on a gel, a single band was found at approximately
60-65 kDa (inset of Fig. 2). This band was eluted (see
"Experimental Procedures") and rerun again on a gel (Fig.
3), both under denaturing and
nondenaturing conditions. Again, only one band was detectable on
silver-stained gels.
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Identification of the Growth Factor as the Granulin/Epithelin
Precursor--
The active fraction was subjected to a number of
analyses, as described under "Experimental Procedures." An aliquot
of the protein preparation was analyzed by direct Edman degradation, because analysis by SDS-PAGE suggested a purity level of 95% for the
growth factor; however, no sequence information was obtained (an
indication of protein blockage). A new and rapid method for the
identification of proteins involved high accuracy peptide mass mapping
using delayed ion extraction MALDI time-of-flight-TOF MS, followed by
protein data base searching with the peptide masses (24). The protein
band shown in Fig. 3 was digested with endoproteinase Lys-C, and an
aliquot of the peptide mixture was used to determine the accurate
molecular masses (generally <30 parts/million) by delayed ion
extraction MALDI-TOF MS. The molecular masses of the peptides were used
to search an in-house nondegenerate protein data base, and the protein
was identified as the rat granulin/epithelin precursor. Table
I compares the regions of the
epithelin/granulin precursor identified by delayed ion extraction MALDI
reflectron TOF MS with the mouse protein and human protein, both
predicted from their respective cDNA. The sequences obtained by us
showed 100% homology with the rat granulin/epithelin, 87% homology to the mouse protein, and 72% homology to the human protein. The growth
factor, it should be noted, was purified from BRL-3A cells that are of
rat origin. The sequences obtained from our band cover 179 of 589 amino
acids or 30.4% of the rat granulin precursor. It seems reasonable to
conclude that at least one of the growth factors in the BRL-3A
conditioned medium that stimulates the growth of R cells
is the granulin/epithelin precursor.
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IRS-1 and MAP Kinase Activation--
We have conducted preliminary
experiments on the possible pathways used by the granulin/epithelin
precursor to induce DNA synthesis in R cells. To
circumscribe the problem, we have investigated the activation (tyrosyl
phosphorylation) of IRS-1 as well as MAP kinase activation by the
granulin/epithelin precursor, following the same procedures outlined
above. These experiments are shown in Fig.
5 (A, B, and
C). IRS-1 tyrosyl phosphorylation is not increased by
stimulation with the granulin/epithelin precursor (Fig. 5A). There is a faint band of phosphorylated IRS-1, but this is present also
in unstimulated cells. We have already noticed before a slight activation of IRS-1 in cells attached to the substratum (10, 11),
probably because of the known interaction of IRS-1 with other cellular
components. Fig. 5B shows the levels of IRS-1 protein in the
immunoprecipitates. We conclude that stimulation with the granulin/epithelin precursor has no effect on tyrosyl phosphorylation levels of IRS-1. This is not the case with MAP kinases, which are
sharply activated by the granulin/epithelin precursor (Fig. 5C), indicating that this growth factor eventually connects
with the MAP kinase pathway in stimulating DNA synthesis.
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DISCUSSION |
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We have purified, partially sequenced, and identified a
polypeptide from BRL-3A conditioned medium that stimulates DNA
synthesis (and growth in number) of R cells that are null
for the IGF-IR (6, 7). This growth factor has been identified as the
granulin/epithelin precursor (14-16). The identification of the
granulin/epithelin precursor as a growth factor that bypasses the
requirement for a functional IGF-IR is of considerable interest for
several reasons: 1) The IGF-IR plays a major role in the growth of
cells, both in vivo and in vitro (see
Introduction), but clearly its requirement is not an absolute one,
because it can be circumvented by a single purified growth factor. 2)
Although epithelins are produced also by normal tissues (see below), in
cells in culture they are predominantly expressed by transformed cell
lines that can grow in serum-free medium. This raises the question
whether the expression of epithelins may also be involved in
transformation. 3) The IGF-IR shares with other growth factor receptors
the main mitogenic pathway (commonly referred to as the ras
pathway). However, the discovery of a purified growth factor that
bypasses this pathway opens the possibility of identifying a new
mitogenic pathway that is presumably ras-independent (7). 4)
One can also predict the usefulness of the granulin/epithelin precursor
in studies dealing with the inhibitory action of IGF-binding proteins,
because stimulation of cell proliferation could be achieved in the
absence of IGFs.
Epithelins were originally purified from rat kidney as two small single
chain peptides (approximately 6 kDa) containing about 20% cysteine
(14). Epithelin1 stimulated the proliferation of murine keratinocytes,
whereas epithelin2 had inhibitory potential (14, 25). At about the same
time, the same peptides were isolated from leukocytes and termed
granulins (26). Subsequently, Bhandari et al. (15) cloned
the granulin precursor cDNA and reported that the prepropeptide of
the human granulins is a 593-residue glycoprotein containing seven
tandem repeats of the 12-cysteine granulin domain. The human
granulin/epithelin gene codes for at least four small epithelin
peptides (27). In addition to granulin A and B, an additional peptide,
granulin F, which had never been isolated before but whose primary
structure is known on the basis of the cDNA sequence, was isolated
from human urine (28). The rat granulin precursor codes for 589 amino
acid residues (BRL-3A cells are from rat liver) and is 72% homologous
to the human granulin precursor and 87% homologous to the mouse
granulin precursor (16, 25). The primary structure of the epithelins
has been reported by Belcourt et al. (29). It has also been
reported that the granulin/epithelin protein motif has an unusual
structure consisting of a parallel stack of -hairpins stapled
together by six disulfide bonds (30). The granulin gene has an unusual
genomic structure containing 12 exons interrupted by 11 introns mapped
to chromosome 17 in human and 13 exons interrupted by 12 introns mapped
to chromosome 11 in mouse, respectively (15, 31, 32). A putative
promoter has also been characterized at the 5' end of the granulin gene (33). The granulin gene is conserved widely in species, suggesting a
possibly widespread growth regulatory function (30).
Although granulin/epithelins have been usually tested as the small
peptides, Zhou et al. (34) purified the precursor protein and showed that it was, by itself, a mitogen for cells in culture. We
are now showing that the granulin/epithelin precursor is mitogenic per
se, in fact that it can stimulate DNA synthesis (and growth, see Ref.
9) in serum-free medium in R cells that are known to be
refractory to stimulation by other purified growth factors, singly or
in combination. Interestingly, Zhou et al. (34) purified the
granulin/epithelin precursor from an "insulin-independent" cell
line. Because insulin in tissue cultures is generally used at
concentrations that activate the IGF-IR (35), their observation is an
indirect confirmation of our observation that the granulin/epithelin
precursor bypasses the requirement for the IGF-IR. The stimulatory
activity of the precursor, in respect to the small epithelins, may be
due to the fact that the small epithelins are a mixture of stimulatory
and inhibitory peptides (14, 25).
Our active polypeptide was inactivated by a denaturing gel. This was not surprising, given the large number of cysteine residues in the sequence (see above); furthermore, structural evidence indicated the importance of proper formation of intra-chain disulfide bonds of the granulin/epithelin (30). Finally, epithelin-binding sites have been found in human breast carcinoma cells (36), but to our knowledge, no further information on a putative receptor has been made available. A 170-175-kDa protein has been reported (37) that binds the epithelial-type transforming growth factor (TGFe), and this growth factor is partially homologous to, but distinct from, the granulin/epithelins (33, 38). This TGFe-specific receptor with a size of approximately 170-175 kDa (37) is presumably different from the putative receptor for granulin/epithelin, a membrane protein of 140-145 kDa (36).
It is worth mentioning that SDS-PAGE of proteins in fractions 34 and 35 corresponding to the first active peak in Fig. 2 showed a single band
of about 25-28 kDa by silver stain (data not shown). Whether it is a
TGFe or a processed granulin/epithelin or another factor remains to be
established. BRL-3A conditioned medium is rich in growth-stimulating
activities (39-41), including IGF-II, originally identified as
multiplication stimulating activity (17). IGF-II, however, cannot
stimulate DNA synthesis in R cells, even at
concentrations up to 200 ng/ml (10), and, in addition, the size of
IGF-II is much smaller than the size of the growth factor we have
purified. It is likely that there are other growth factors in the
BRL-3A conditioned medium that can stimulate growth of R
cells. The one we have purified and identified is the
granulin/epithelin precursor.
The identification of the granulin/epithelin precursor as a growth
factor that bypasses the requirement for an activated IGF-IR is, in our
opinion, important, both from a basic and an applied point of view (see
above). The IGF-IR is found in many cell types and, as mentioned in the
Introduction, is necessary for the optimal growth of cells in
vivo and in vitro. Furthermore, down-regulation of the
IGF-IR causes massive apoptosis of tumor cells in vivo, whereas overexpression causes transformation and protection from apoptosis (reviewed in Ref. 4). The granulin/epithelin family could
then become a target in those tumor cells that have escaped regulation
by the IGF-IR. At a more basic level, our finding raises some
interesting questions, for instance, whether the granulin/epithelin peptides use and, so to speak, usurp the IGF-IR signaling pathway(s), or whether they induce mitogenicity (and/or transformation) by a
totally different pathway. This problem is being actively investigated, but our preliminary results shown in Fig. 5 clearly indicate that MAP
kinases are activated, but IRS-1 is not. Thus, the pathway stimulated
by the granulin/epithelin precursor does not seem to be dependent on
IRS-1 and is therefore different from the IGF-IR mitogenic pathway,
which is heavily dependent on IRS-1 (reviewed in Ref. 4). On the other
hand, this pathway connects again with one of the main pathways for
stimulation of DNA synthesis, which passes through the MAP kinases. A
considerable amount of evidence has accumulated, indicating a crucial
role of MAP kinases in the stimulation of DNA synthesis by either
growth factors or integrins or simply attachment to the substratum (23,
42-45). Indeed, MAP kinase activation has been reported to increase
transcription from the serum response element (45), which is usually
correlated with stimulation of DNA synthesis. Thus it seems that the
granulin/epithelin precursor bypasses the IRS-1 mitogenic pathway but
eventually joins the mitogenic pathway that is common to many growth
factors and mitogenic stimuli (23, 42-45). The question now is where the granulin/epithelin pathway reinserts itself into the main mitogenic
pathway, downstream from IRS-1. At present, the only clue we have is
that a plasmid expressing the human granulin/epithelin precursor
(courtesy of Dr. Bateman) can make R cells grow in
serum-free medium, a property that, so far, is shared only by
v-src (13). This finding suggests that src may be
involved in granulin/epithelin signaling. The connection between c-src and MAP kinases has been elucidated recently by
Schlaepfer et al. (23), and the src pathway may
therefore be a prime candidate for further studies. However, the
complexities of the mitogenic signaling pathways is such that several
options must be kept open, especially in view of the fact that no
receptor for the granulin/epithelin precursor has yet been cloned. The
R
cells expressing the granulin/epithelin precursor are
now being investigated in detail for their growth phenotype, including
transformation and ability to protect cells from apoptosis and the
ability to process the precursor and their signaling potentials.
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FOOTNOTES |
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* This work is supported by Grant AG 00378 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Kimmel Cancer
Center, Thomas Jefferson University, 233 S. 10th Street,
624 BLSB, Philadelphia, PA 19107. Tel.: 215-503-4507; Fax:
215-923-0249; E-mail: r_baserga{at}lac.jci.tju.edu.
The abbreviations used are: IGF, insulin-like growth factor; IGF-IR, type 1 IGF receptor; TGF, transforming growth factor; TGFe, epithelial-type TGF; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MS, mass spectrometry; PAGE, polyacrylamide gel electorphoresis; MAP, mitogen-activated protein.
2 M. Mann (1996) peptide search software for Apple Macintosh computers (available via anonymous ftp at ftp://mac-mannb.embl-heidelberg.de/saturn/pub/software).
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REFERENCES |
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