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J. Biol. Chem., Vol. 276, Issue 32, 30183-30187, August 10, 2001
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From the Department of Medicine, Thyroid Division, Brigham
and Women's Hospital and Harvard Medical School, Boston, Massachusetts
02115 and
Received for publication, June 13, 2001, and in revised form, June 22, 2001
Types 1 and 3 iodothyronine
deiodinases are known to be selenocysteine-containing enzymes. Although
a putative human type 2 iodothyronine deiodinase (D2) gene
(hDio2) encoding a similar selenoprotein has been
identified, basal D2 activity is not selenium (Se)-dependent nor has D2 been labeled with
75Se. A human mesothelioma cell line (MSTO-211H) has
recently been shown to have ~40-fold higher levels of
hDio2 mRNA than mesothelial cells. Mesothelioma cell
lysates activate thyroxine (T4) to
3,5,3'-triiodothyronine with typical characteristics of D2 such
as low Km (T4), 1.3 nM,
resistance to propylthiouracil, and a short half-life (~30 min). D2
activity is ~30-fold higher in Se-supplemented than in Se-depleted
medium. An antiserum prepared against a peptide deduced from the
Dio2 mRNA sequence precipitates a 75Se
protein of the predicted 31-kDa size from 75Se-labeled
mesothelioma cells. Bromoadenosine 3'5' cyclic monophosphate increases
D2 activity and 75Se-p31 ~2.5-fold whereas substrate
(T4) reduces both D2 activity and 75Se-p31
~2-3-fold. MG132 or lactacystin (10 µM), inhibitors of the proteasome pathway by which D2 is degraded, increase both D2
activity and 75Se-p31 3-4-fold and prevent the loss of D2
activity during cycloheximide or substrate (T4) exposure.
Immunocytochemical studies with affinity-purified anti-hD2 antibody
show a Se-dependent increase in immunofluorescence. Thus,
human D2 is encoded by hDio2 and is a member of the
selenodeiodinase family accounting for its highly catalytic efficiency
in T4 activation.
A group of three specific deiodinases monodeiodinate thyroxine
(T4)1 to
3,5,3'-triiodothyronine (T3), the active thyroid
hormone, or 3,3',5'-triiodothyronine, an inactive metabolite. The first of these enzymes to be cloned was the type 1 iodothyronine deiodinase (D1), which revealed a rare structural characteristic,
i.e. the presence of the selenocysteine
(Sec) codon, UGA, in the active center of the enzyme conferring an
~200-fold higher catalytic efficiency than sulfur in the deiodination
reaction (1). It also revealed a requirement for a stem loop structure
in the 3'-untranslated region, the Sec insertion sequence
(SECIS) element (2), which is found in all eukaryotic selenoprotein
mRNAs described to date (3).
The most recently cloned deiodinase is the type 2 (D2), a
propylthiouracil (PTU)-resistant, low Km
(T4) obligate outer ring deiodinase that catalyzes
T4 to T3 conversion (4, 5). The major open
reading frame of the hD2 cDNA encodes a putative ~31-kDa protein
with high similarity of the Sec-containing active center of the
putative D2 enzyme with those of D1 and the other member of this group,
type 3 iodothyronine deiodinase (D3) (6). Furthermore, a SECIS element
has been identified in the extreme 3'-untranslated region of both the
human and highly homologous chicken Dio2 genes (7,
8). Human, mouse, rat, chicken, and frog D2 all contain a putative
in-frame UGA codon in the deduced amino acid sequence of the active
center (position 133 in hD2) and therefore all are considered to be
selenoenzymes (8).
Despite this evidence, there is vigorous disagreement that D2 is a
selenoprotein, with some investigators claiming that Dio2 encodes a "virtual" or artificial selenoprotein that is not
expressed in humans or rats (9-14). Three major arguments support this
position, namely 1) the failure of Se deficiency to reduce D2 catalytic activity either in vivo or in vitro (9, 15, 16),
2) the inability to identify a 75Se-labeled protein of the
expected size in cells expressing D2 (13), and 3) the inability to
identify immunoreactive protein by Western analysis,
immunoprecipitation (IP), or immunocytochemistry using antibodies
prepared against peptides deduced from the sequence of the putative D2
mRNA (4). An alternative scenario put forward is that D2 is a large
protein complex (200 kDa) containing one or more 29-kDa
substrate-binding subunits (p29), an ~60-kDa cAMP-induced activation
protein, and one or more catalytic subunits (12). Rat p29 was
identified in astrocytes by
N-bromoacetyl-T4 labeling (17-19), has
no enzymatic activity, and is highly similar to Dickkopf-3 (20).
Although many of these apparent discrepancies could be explained by low
expression of a highly efficient enzyme, reservations as to the
identity of D2 are expressed even in recent reviews of the subject (21,
22).
A mesothelioma cell line, MSTO-211H, was recently shown to express
large amounts of Dio2 mRNA by microarray analysis
(accession number U53506), offering a potential system to
resolve the issue of D2 identity (23).
Reagents--
MG132 and lactacystin were obtained from
Calbiochem (La Jolla, CA) and dissolved in Me2SO.
MG132 is a reversible proteasome inhibitor. Lactacystin blocks
proteasome activity by targeting the catalytic Cell Culture and D2 Activity--
Mesothelioma (MSTO-211H) and
mesothelial (MeT-5A) cell lines were obtained from American Type
Culture Collection (ATCC; Manassas, VA) and made available through
Dr. Rihn. Cells were plated in 60-mm dishes and grown until confluence
in RPMI or M-199, respectively, and supplemented with 0-10% fetal
bovine serum (FBS). Human embryonic kidney epithelial cells (HEK-293)
cells were incubated in Dulbecco's modified Eagle's medium with 0 or
10% fetal bovine serum. Each experiment was performed with duplicate
dishes for each condition, and control plates contained the respective
vehicles, 0.2% Me2SO and/or 0.6 mM NaOH. At
the appropriate times, cells were harvested and processed for D2
activity in the presence of 1 mM PTU as described previously (24). The results are reported as fmol T4
deiodinated/mg protein·min ± S.D.
Procedures for Tranfections--
In some experiments D2 was
transiently expressed in HEK-293. Cells were transfected by the
CaPO4 method as described previously (25) with plasmids
containing wild type D2 (KD2-SelP), a D2 mutant in which the Sec-133
(but not Sec-266) was replaced by Cys (CysD2), or a CysD2 in which the
SECIS element was deleted (CysD2 75Se Incorporation and D2 IP--
This was performed
as described previously (25) after cells were labeled with 4-6 µCi
of Na2[75Se]O3/dish. The lysis
buffer contained 25 mM Tris-HCl (pH 7.4), 300 mM NaCl, 1 mM CaCl2, 0.5% Triton
X-100, 10 µg/ml leupeptin, 0.2 units/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride. The 2,000-rpm supernatant of each cell
lysate was incubated for 16 h at 4 °C with D2 rabbit antisera
85254 at final dilution of 1:100 (25). 30 µl of protein G
plus/protein A-agarose solution were added per tube and incubated under
slow agitation for 2 h at 4 °C. The IP pellets were washed once
with lysis buffer and once with 25 mM Tris-HCl (pH 7.4),
140 mM NaCl, 1 mM CaCl2. Pellets were re-suspended in sample loading buffer and analyzed in 12% SDS-PAGE. In some experiments IP was processed in unlabeled cells to
allow measurement of D2 activity.
Affinity Purification of D2 Antiserum--
For the
affinity purification of the antiserum 45618 (25) the keyhole
limpet hemocyanin conjugate of the hD2 peptide (EVRSWLEKNFSKR; residues
253 to 265) used to immunize the rabbits was coupled to agarose
resin using Amino-Link agarose gel (Pierce) following the
manufacture's instructions. The column was regenerated by washing with
3 M sodium acetate (pH 4.5) and equilibrated with phosphate-buffered saline (PBS) prior to each cycle. The D2 antiserum (45618) was passed through the column five times and then washed 10 times with PBS. Bound antibodies were eluted with 3 M
sodium acetate (pH 4.5), and eluates were dialyzed against PBS at
4 °C for 2 days. This affinity-purified antiserum (AP-45618; 1:400) was used in the immunocytochemical and Western analysis of MSTO-211H or
MeT-5A cells as described (26).
Immunocytochemistry and Confocal Analysis--
This was
performed as described previously (27). MSTO-211H and MeT-5A cells were
grown directly in glass slides for 24 h, fixed with 3.7%
paraformaldehyde in PBS (pH 7.4), permeabilized with 0.5% Triton X-100
in PBS for 10 min, and then blocked for 30 min with 1.0% anti-goat
serum, 1.0% bovine serum albumin in PBS. Affinity-purified rabbit D2
antibody (AP-45618) was used at 1:500 followed by incubation with 1.25 µg/ml goat anti-rabbit fluorescein isothiocyanate (Molecular Probes,
Eugene OR). In the co-localization experiments, the affinity-purified
anti-goat GRP78/BiP (dilution 1:20) antibody (Research Diagnostics,
Inc., Flanders, NJ) was used simultaneously with anti-D2 antibody. This
was followed by incubation with donkey anti-goat rhodamine
F(ab')2 fragment (Jackson ImmunoResearch Laboratories, West
Grove, PA) and with goat anti-rabbit fluorescein isothiocyanate.
Characterization of hD2 in MSTO-211H Cells and the Effects of
Se--
Mesothelioma (MSTO-211H) cell sonicate converts T4
to T3 with kinetic properties typical of native D2, namely
a Km for T4 of ~1.0 nM and
insensitivity to inhibition by 1 mM PTU as shown in the
Lineweaver-Burk plot (Fig.
1A). Neither D1 nor D3
activities were detected nor did the transformed normal mesothelial cells (MeT-5A) express D2 activity (not shown). D2 activity in MSTO-211H cells was highly Se-dependent. Basal activity was
decreased 4-fold by reducing the concentration of FBS containing
endogenous selenoproteins (Fig. 1B). That these changes are
specifically caused by Se deficiency and not by other components in FBS
is evident from the fact that Se supplementation progressively
increased D2 activity at each concentration of FBS, from 2-3-fold at
10% FBS up to ~30-fold when no FBS was present (Fig. 1B).
The Lineweaver-Burk plots from cells incubated in the absence of FBS
with or without 100 nM Na2SeO3 for
24 h shows an ~16-fold increase in Vmax
with no change in the Km (Fig. 1A). A
dose-response curve of D2 activity versus
Na2SeO3 concentration indicated that the maximum stimulatory effect is obtained at 100 nM. A
time-response curve showed that an effect of Se is present after 2 h, but the full effect requires 8 h (not shown). Similar effects
of Se to increase D2 activity were observed in HEK-293 cells
transiently expressing Sec-encoding D2 mRNA. Exposure to 100 nM Na2SeO3 for 24 h increased
D2 activity by ~10-fold suggesting this effect is primarily
post-transcriptional (Fig. 1B).
To confirm that the MSTO-211H cells express a selenoprotein containing
a deduced D2 epitope, cells were labeled with
Na2[75Se]O3 for 24 h and
processed for D2 IP using an anti-hD2 peptide antiserum (85254) (25).
Mesothelial (MeT-5A) cells were used as negative controls. Four
75Se-labeled bands of ~70, 58, 25, and 18 kDa (but not 31 kDa) are present in the SDS-PAGE of total cell lysates of MSTO-211H or MeT-5A cells (Fig. 1C, b). These
75Se-labeled proteins are increased by incubation with 100 nM Na2SeO3 in MSTO-211H cells but
are decreased in MeT-5A (Fig. 1C, b). SDS-PAGE analysis of the anti-D2 IP of the MSTO-211H cell lysate revealed an
~31-kDa 75Se-labeled doublet band (p31) of the predicted
size of hD2, the intensity of which is increased ~2-fold in the
presence of 100 nM Na2SeO3 (Fig.
1C, c and d). A doublet is seen when
hD2 cDNA is transiently expressed because of the presence of a
second UGA at the 3'-end of the open reading frame that can be
translated either as a Sec or as a stop codon (28). The content
of all other 75Se-labeled proteins is reduced in the
anti-D2 IP pellets (Fig. 1C, c). IP of
non-labeled cell lysates reduced D2 activity ~20%, and ~ Fluctuation in 75Se-p31 Levels Parallel D2 Activity in
MSTO-211H Cells--
The hD2 gene contains a canonical cAMP-response
element-binding protein-binding site about 90 base pairs 5' to the most
5'-transcription start site (29). D2 activity increases in cells
treated for 6 h with 8-Br-cAMP in a dose-dependent
fashion (Fig. 2A). To test whether there is a correlation between 8-Br-cAMP-induced D2 activity and p31, cells treated with 1 mM 8-Br-cAMP were labeled
with 75Se and processed for D2 IP. The intensity of the
75Se-p31 band increased ~2.5-fold in 8-Br-cAMP-treated
cells (Fig. 2F). No effects of insulin (0.1-5
µM) or tumor necrosis factor-
Both endogenous and transiently expressed D2 have a short-life (<1 h)
that is reduced further by exposure to substrates such as
3,3',5'-triiodothyronine or T4 (24, 25, 30-32). This is explained by ubiquitin conjugation and subsequent proteasomal degradation of the ubiquitinated D2 (25, 26). Treatment of cells with
100 µM CX for 1 h resulted in a rapid loss of D2
activity, compatible with a D2 half-life of <30 min (Fig.
2B). The decrease in D2 activity was prevented by the
proteasome inhibitor, MG132 (10 µM) (Fig. 2B).
Furthermore, exposure to MG132 increased D2 activity up to ~4-fold,
with similar results obtained with 10 µM lactacystin
(Fig. 2C). Remarkably, anti-D2 precipitable
75Se-p31, but not other labeled proteins, was also
specifically increased by treatment with MG132 (Fig. 2F).
Exposure to substrate (0.01-10 µM T4 in 10%
FBS) for 1 h caused a concentration-dependent reduction in D2 activity of up to 10-fold (Fig. 2D), which
again was blocked by MG132 (Fig. 2E). This was accompanied
by a reduction in the anti-D2 precipitable 75Se-p31 (Fig.
2F).
MSTO-211H and MeT-5A cells were also analyzed by
immunocytochemistry using an affinity-purified anti-D2 antibody
(AP-45618). In MSTO-211H cells incubated in serum-free medium
for 24 h, the cytoplasm showed mild staining for D2 (Fig.
3A). Treatment with 100 nM Na2SeO3 for 24 h
substantially increased the intensity of the staining without changing
the distribution pattern (Fig. 3B). No fluorescence was
detected in MSTO-211H cells incubated with secondary antibody alone
(Fig. 3C) or in Na2SeO3-treated MeT-5A cells incubated with affinity-purified anti-D2 antibody (Fig.
3D). To study the subcellular distribution of endogenously expressed D2, MSTO-211H cells were stained with D2 antibody (Fig. 3F) and co-stained with antibody raised against the
endoplasmic reticulum resident protein GRP78/BiP (Fig. 3G),
shown previously to colocalize with transiently expressed D2 in HEK-293
and neuroblastoma cells (27). Co-localization was confirmed by
superimposition of confocal images (Fig. 3H).
The Se-supplemented MSTO-211H cells have the highest D2 activity
reported to date in a human tissue (~140 fmol/min/mg protein), ~40% higher than in Graves' thyroid tissue (33). This high D2 expression permits the unequivocal demonstration that endogenous hD2 is
a selenoprotein encoded by the Dio2 gene (5). The present results satisfy a number of important criteria establishing the identity of hD2 as the product of the Dio2 gene.
Furthermore, they allow confirmation that the endogenous human enzyme
behaves as predicted from a number of studies using transiently
expressed protein.
The first important criterion to establish D2 as a selenoprotein is to
demonstrate its Se dependence. Past studies showed that the elevated D2
in cerebral cortex of thyroidectomized rats is not reduced by Se
deficiency (15) and that astroglial cells depleted of Se for 3-5 days
did not have lower basal D2 activity (9). Although there was about 50%
reduction of the D2 activity in cAMP-treated primary astrocyte
cultures after 7 days of Se deprivation, basal D2 activity was still
not affected (34). However, the central nervous system is well known to
retain Se with high efficiency, which could explain why D2 synthesis is not impaired by Se deficiency (35).
In MSTO-211H cells, on the other hand, Se depletion for 24 h
reduced basal D2 activity by ~4-fold. This decrease in D2 activity was specifically reversed by Se supplementation (Fig. 1, A
and B). In fact, addition of Se increased D2 activity by
~30-fold in Se-depleted cells, in a dose- and
time-dependent fashion, a phenomenon that also occurs in
HEK-293 cells transiently expressing D2 (Fig. 1, A and
B). Selenium depletion of MSTO-211H cells also markedly
reduces the levels of another selenoprotein, glutathione peroxidase
(36).
A second criterion met by the present studies is the 75Se
labeling and immunological recognition of D2. A recent study could not
identify the 75Se-D2 protein by IP using antibodies that
were raised against the COOH terminus of full-length rat D2 or against
the catalytic core of the enzyme (13). This is not the case in the
present studies (see Fig. 1C and Fig. 2F). Aside
from potential differences in the antisera per se, which
might be an issue, the D2 activity in rat tissue sonicates or
stimulated primary astrocyte cultures are only ~10% of that in
MSTO-211 cells. Because of the higher D2 expression, we can demonstrate
IP of D2 activity, as well as of a 75Se-labeled protein of
the predicted size (31 kDa) from MSTO-211H cells (Fig. 1C).
This is the first demonstration of endogenous 75Se-D2. The
75Se-labeled 31-kDa protein is increased by
Na2SeO3, mirroring changes in D2 activity (Fig.
1C). A correlation between changes in D2 activity and the
amount of 75Se-p31 is also evident during treatment with
proteasome inhibitors (see below), 8-Br-cAMP, or D2 substrate (Fig.
2).
Additional immunological identity criteria were obtained by
immunocytochemical studies using the affinity-purified anti-hD2 antibody. Staining was only present in MSTO-211H cells and was markedly
increased by Se supplementation (Fig. 3, A-D). Furthermore, D2 co-localized with the endoplasmic reticulum-specific marker BiP as
found previously in HEK-293 and neuroblastoma cells transiently expressing hD2 (Fig. 3, E-H) (27). Despite successful IP of the D2 protein and activity, we were unable to visualize D2 by Western
blot analysis presumably because of low antibody affinity for denatured
protein, low D2 expression, or both.
A short half-life and the acceleration of endogenous D2 degradation by
exposure of cells to substrates observed in pituitary tumor cells and
in cells transiently expressing D2 are reproduced in these experiments.
A short half-life (<1 h) is characteristic of D2 in all cells studied
(11, 24, 31) and is explained by the fact that D2 is the target of
selective ubiquitination and proteolysis by the proteasomal system
(24-26). This is also the case in MSTO-211H cells, in which D2
activity and 75Se-D2 protein levels were extremely
sensitive to inhibitors of the proteasomal pathway (Fig. 2,
B-E). It is remarkable that in GH4C1 rat pituitary tumor
and transfected HEK-293 cells, treatment with proteasomal inhibitors
for 1 h increases D2 activity by only ~20% (24, 25) whereas in
the present study a similar treatment resulted in a 3-4-fold increase
in activity (Fig. 2C). This can be explained by a more rapid
turnover of the enzyme in mesothelioma cells. Furthermore, as in
pituitary tumor cells and transfected HEK-293 cells, exposure to
T4, the preferred D2 substrate, accelerates the turnover,
increasing ubiquitination and subsequent proteolysis, a phenomenon that
can be specifically inhibited by treatment with proteasomal inhibitors
(Fig. 2, D-E). These results demonstrate that the targeting
of D2 to the ubiquitin-proteasomal system and the acceleration of its
degradation are characteristic of this protein, regardless of the cell
type in which it is present or whether it is transiently expressed or endogenous.
In conclusion, the evidence developed in the present study
unequivocally establishes that endogenous hD2 is a short-lived selenoprotein, the product of the human Dio2 gene, as well
as the 90-95% homologous frog, chicken, mouse, and rat proteins. These findings are also consistent with preliminary data reported recently that targeted disruption of the mouse Dio2 gene
eliminates D2 activity in brain, pituitary gland, and brown adipose
tissue (37). From a broader perspective it is interesting to note that this is the second example of high levels of a selenodeiodinase in
human tumor cells derived from a tissue that normally does not express
deiodinase, the other being the high levels of D3 in hemangiomas (38).
It will be of interest to determine whether primary mesotheliomas also
express high levels of D2 and what, if any, effects of increased rate
of T4 to T3 conversion might have in these cells.
We thank Dr. Stephen Huang for
assaying MSTO-211H cell lysates for D3 activity.
*
This work was supported by National Institutes of Health
Research Grants DK36256 and DK58538.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: Brigham and Women's
Hospital, 77 Ave. Louis Pasteur, HIM Bldg. 550, Boston, MA 02115. Tel.:
617-525-5158; Fax: 617-731-4718; E-mail:
abianco@rics.bwh.harvard.edu.
Published, JBC Papers in Press, June 25, 2001, DOI 10.1074/jbc.C100325200
The abbreviations used are:
T4, thyroxine;
T3, 3,5,3'-triiodothyronine;
D1, type 1 iodothyronine deiodinase;
Sec, selenocysteine;
D2, type 2 iodothyronine deiodinase;
PTU, propylthiouracil;
h, human;
D3, type 3 iodothyronine deiodinase;
IP, immunoprecipitation;
CX, cycloheximide;
8-Br-cAMP, 8-bromoadenosine
3'5' cyclic monophosphate;
FBS, fetal bovine serum;
HEK, human
embryonic kidney;
PAGE, polyacrylamide gel electrophoresis;
PBS, phosphate-buffered saline;
SECIS, Sec insertion sequence.
The Human Type 2 Iodothyronine Deiodinase Is a Selenoprotein
Highly Expressed in a Mesothelioma Cell Line*
,, Institut National de Recherche et de Securite,
54501 Vandoeuvre Cedex, France
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit and
covalently inhibiting the chymotrypsin- and trypsin-like activities.
T4, cycloheximide (CX), and 8-bromoadenosine 3'5' cyclic
monophosphate (8-Br-cAMP) were from Sigma and were dissolved in 40 mM NaOH (T4) or
Me2SO. Protein G plus/protein A-agarose solution was
obtained from Oncogene Research Products (Boston, MA). Outer
ring-labeled [125I]T4 (specific activity,
4400 Ci/mmol) was from PerkinElmer Life Sciences.
Na2[75Se]O3 was kindly provided
by the University of Missouri Research Reactor, courtesy of
Drs. Marla Berry and Dolph L. Hatfield. All other reagents were of
analytical grade.
Xba).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (37K):
[in a new window]
Fig. 1.
D2 activity is present in MSTO-211H cells and
is influenced by Se concentration in the media. A,
Lineweaver-Burk plot of the D2 substrate saturation curve using lysates
of MSTO-211H cells incubated in serum-free media ± 100 nM Na2SeO3 for 24 h.
prot, protein. B, D2 activity in lysates of
MSTO-211H or HEK-293 cells transiently expressing hD2 incubated in
media containing 1, 5, or 10% FBS ± 100 nM
Na2SeO3 for 24 h. C,
autoradiographs of SDS-PAGE analyses of 75Se-labeled cell
lysates. a, IP pellets of HEK-293 cells transfected with
CysD2. No doublet is seen here, because this cDNA contains a
Sec133Cys mutation, and the 75Se labeling is
restricted to Sec-266. CysD2 xba, which lacks a SECIS element, was
used as a negative control. The film was exposed for 10 days.
b, total cell lysates of MSTO-211 and MeT-5A cells incubated
in media containing 10% FBS ± Na2SeO3.
The film was exposed for 1 week. c, IP pellets from the same
samples shown in b. The film was exposed for 3 weeks.
d, detail of 75Se-p31 doublet.
75Se-p31 was successfully visualized in four similar but
separate experiments. D, D2 activity in supernatants
(SN) of MSTO-211H cell lysates after IP with preimmune serum
or anti-D2 antiserum and in the IP pellets. In panels A,
B, and D, values are the mean ± S.D. of two
cell plates, and each experiment was repeated twice.
(0.01-10 ng/ml) on D2
activity were detected (data not shown).
View larger version (34K):
[in a new window]
Fig. 2.
D2 activity correlates with
75Se-p31 levels in MSTO-211H cells. Cells were
incubated with agents known to alter D2 activity. 75Se-p31
levels were determined by IP. A, cells incubated with 0, 0.3, or 1 mM 8-Br-cAMP for 6 h. prot,
protein. B, cells incubated with 0.2% Me2SO
(vehicle), 100 µM CX, or 10 µM MG132 plus
100 µM CX for 40 min. MG132 was added 20 min before CX.
C, cells incubated with Me2SO, 10 µM MG132, or 10 µM lactacystin
(Lacta) for 4 h. D, cells incubated with 0, 0.01, 0.1, 1, or 10 µM T4 for 90 min in the
presence of 10% FBS. E, cells incubated with 15 µM T4 ± 10 µM MG132 for 2 h. F, autoradiograph of SDS-PAGE analysis of IP pellets
obtained from 75Se-labeled-MSTO-211H cells treated as in
A (1 mM 8-Br-cAMP), C (10 µM MG132), and E (10 µM
T4). Film was exposed for 3 weeks. This experiment was
repeated twice with identical results. In panels
A-E values are the mean ± S.D. of two cell
plates, and each experiment was repeated two to five times.
View larger version (43K):
[in a new window]
Fig. 3.
Immunocytochemical analysis of D2 in
MSTO-211H cells. Confocal microscopic localization of D2 in
MSTO-211H cells grown in the absence (a) or presence
(b) of 100 nM Na2SeO3
(± S.E.). No signal was detected in MSTO-211H cells stained with
secondary antibody only (c) or in MeT-5A cells incubated
with anti-D2 antibody (d). Panel e is a phase
contrast of a typical MSTO-211H cell co-stained with anti-D2 antibody
(f) and GRP78/BiP protein (g). The
superimposition of the same fields is shown in h, where the
inset represents the distribution spectrum of image pixels. Cell types
are indicated in the upper left corner. Bar = 50 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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