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Mol Cell Biol, March 1998, p. 1236-1247, Vol. 18, No. 3
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Overexpression of the Nucleoporin CAN/NUP214
Induces Growth Arrest, Nucleocytoplasmic Transport Defects,
and Apoptosis
Judith
Boer,
Jacqueline
Bonten-Surtel, and
Gerard
Grosveld*
Department of Genetics, St. Jude Children's
Research Hospital, Memphis, Tennessee
Received 2 September 1997/Returned for modification 19 October
1997/Accepted 2 December 1997
 |
ABSTRACT |
The human CAN gene was first identified as a target of
t(6;9)(p23;q34), associated with acute myeloid leukemia and
myelodysplastic syndrome, which results in the expression of a
DEK-CAN fusion gene. CAN, also called NUP214, is a nuclear
pore complex (NPC) protein that contains multiple FG-peptide sequence
motifs. It interacts at the NPC with at least two other proteins, the
nucleoporin NUP88 and hCRM1 (exportin 1), which was recently shown to
function as a nuclear export receptor. Depletion of CAN in knockout
mouse embryonic cells results in cell cycle arrest in G2,
followed by inhibition of nuclear protein import and a block of mRNA
export. We overexpressed CAN and DEK-CAN in U937 myeloid precursor
cells. DEK-CAN expression did not interfere with terminal myeloid
differentiation of U937 cells, whereas CAN-overexpressing cells
arrested in G0, accumulated mRNA in their nuclei, and died
in an apoptotic manner. Interestingly, we found that hCRM1 and import
factor p97/importin 
colocalized with the ectopically expressed CAN
protein, resulting in depletion of both factors from the NPC.
Overexpression of the C-terminal FG-repeat region of CAN, which
contains the binding site for hCRM1, caused sequestering of hCRM1 in
the nucleoplasm and was sufficient to inhibit cell growth and to induce
apoptosis. These results confirm that CAN plays a crucial role in
nucleocytoplasmic transport and imply an essential role for hCRM1 in
cell growth and survival.
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INTRODUCTION |
The recurrent chromosome
translocation (6;9)(p23;q34), found in acute myeloid leukemia and
myelodysplastic syndrome, fuses the coding regions of two genes,
DEK and CAN (52). The resulting DEK-CAN mRNA encodes an in-frame chimeric protein that
contains almost the entire DEK protein linked to the C-terminal
two-thirds of CAN. DEK is a nuclear DNA-binding protein
(16). CAN, also called NUP214, is a nuclear pore complex
(NPC) component, or nucleoporin, and contains NPC protein-specific
FG-repeat sequences (12, 30). Its deletion in mouse embryos
results in cell cycle arrest in G2 followed by a block in
mRNA export and inhibition of nuclear protein import (49).
The central region of CAN contains two predicted coiled-coil domains
and anchors the protein to the NPC. Approximately the same domain binds
NUP88, a novel NPC component of 88 kDa (13, 14). The
translocation breakpoint in CAN lies in the middle of this region, and
in the DEK-CAN fusion protein both nuclear envelope localization and
NUP88 binding are disrupted (13). DEK-CAN is nuclear, and
relocation of the C terminus of CAN from the nuclear envelope to the
nucleus may contribute to the leukemogenic potential of the fusion
protein (12). The C terminus of CAN, consisting of
nucleoporin-specific repeats, can direct the protein to the nucleus in
the absence of the NPC-binding domain (12). This relocation
is mediated by binding of this part of the repeat, which is present in
CAN as well as in DEK-CAN, to hCRM1, a protein of 112 kDa that was
found to shuttle between the NPC and the nucleus (13, 14).
hCRM1 is a member of a newly identified family of NPC-interacting
proteins, which suggested that hCRM1 might function as a
nucleocytoplasmic transport factor (14). Indeed, CRM1 was recently identified as an export receptor for leucine-rich nuclear export signals (NESs) (14a, 46).
The NPC is a supramolecular structure that contains multiple copies of
about 100 different proteins. It mediates bidirectional transport of
macromolecules between the cytoplasm and the nucleus (reviewed in
references 8, 11, 38 and 42).
Karyophilic proteins contain a nuclear localization signal (NLS) that
is recognized by the NLS receptor, also called importin 
, Srp1p, or
karyopherin (1, 2, 22, 35, 54). This complex docks to
the cytoplasmic side of the NPC via p97, synonymous with importin and karyopherin (7, 23, 39). p97 binds to
repeat-containing nucleoporins in vitro and in vivo (27, 31,
39-41). After this initial docking step, the complex is
translocated through the pore via an energy-dependent process that is
mediated by the small nuclear GTPase Ran and its cofactors (33,
34). At the nuclear side of the NPC, the NLS receptor and the
substrate are released into the nucleoplasm while p97 remains bound to
the NPC (23, 36).
Protein and ribonucleoprotein (RNP) export from the nucleus also occurs
via a receptor-mediated, energy-dependent mechanism. NESs, identified
in a number of proteins, are thought to play a role in this process
(for reviews, see references 18, 21, and
28). Leucine-rich NESs can be transported by hCRM1,
and overexpression of hCRM1 was shown to increase the export of human immunodeficiency virus type 1 (HIV-1) genomic mRNA and U small nuclear
RNA (UsnRNA). Importin was recently found to be involved in the
export of capped RNA polymerase II transcripts from the nucleus, and
dissociation of the importin -RNA complex into the cytoplasm is
mediated by importin (20).
Depletion of nuclear pore components often leads to nucleocytoplasmic
transport defects, growth inhibition, and cell death. Such effects have
been demonstrated for several yeast mutants (10, 42), and
recently for CAN/NUP214 in the mouse (49). The yeast studies
have also shown that it is important to maintain the correct relative
stoichiometry of NPC components, since overexpression of some
components severely restricts cell growth (9, 55, 56).
Overexpression of CAN and DEK-CAN in cell lines has proven to be toxic
(12). To address why overexpression of these proteins is
cytotoxic and to study the effects of overexpression on myeloid differentiation, we introduced inducible CAN and DEK-CAN genes into the
human myeloid precursor U937 cells. Expression of the acute myelogenous
leukemia-specific DEK-CAN protein did not affect the differentiation of
U937 cells, whereas overexpression of CAN in U937 cells arrested them
in G0, caused a defect in mRNA export and mislocalization
of hCRM1 and p97, and ultimately led to apoptosis. Overexpression of
the hCRM1-binding domain of CAN resulted in nuclear sequestering of
hCRM1 and was sufficient to inhibit cell growth and to induce cell
death.
| |
MATERIALS AND METHODS |
Expression constructs.
All the expression plasmids used in
this study carry sequences encoding two copies of the influenza virus
hemagglutinin (HA1) tag at the 5' ends of their open reading frames
(12) and are driven by the tetVP16-responsive promoter
(24). Expression of CAN, DEK-CAN, CAN 1-1058, CAN
586-1058, CAN 816-2090, CAN 1864-2090, and CAN 1558-1840 was
directed by plasmids described previously (12, 13). CAN
1140-2090 expression was directed by plasmid pHA1-Can 
1-1139
(12). The pHA1-CAN construct (1140-1340, 1864-1912, 1984-2090) was derived from pHA1-CAN (1140-1340, 1864-2090) by an
in-frame deletion of the fragment between the BamHI sites at positions 5825 and 6044 of the can cDNA. Plasmid pHA1-CAN
1864-2017 was derived by subcloning the region that encodes amino
acids 1864 to 2090 from pHA1-CAN (1-1139, 1341-1863) and
inserting an oligonucleotide containing translational stops in the
PstI site at position 6145 of the can cDNA. A
plasmid containing the murine Bcl-xL cDNA, driven by the
spleen focus-forming virus long terminal repeat, was kindly provided by
G. Nuñez.
Inducible gene expression in U937T cells.
The human
monoblast cell line U937 (47) and its derivatives were
routinely cultured in RPMI 1640 medium supplemented with 10% fetal
bovine serum in a 37°C incubator with 5% CO2.
Transfections were performed by electroporation at 0.17 kV and 960 µF
on a Bio-Rad gene pulser. To generate cell lines inducibly expressing
the proteins of interest, we used a two-step procedure. First, U937
cells were transfected with pUHD/TetVP16Puro
(51), encoding tetVP16 under the control of the
tetVP16-responsive promoter (24), thereby making tetVP16
expression tetracycline repressible and autoregulatory (45).
Transfected cells were selected in 0.5 µg of puromycin per ml in the
presence of 1 µg of tetracycline per ml. Single clones were examined
for tetVP16 expression by RNA dot blotting of cells grown in the
presence or absence of tetracycline. Of the 18 clones examined, 7 showed tetracycline-controlled expression of tetVP16. Second, clone
U937T was selected for subsequent stable transfection with expression
constructs containing HA1-epitope tagged cDNAs under the control of the
tetVP16-responsive promoter. pHA1-CAN was cotransfected with the
neomycin-selectable pMC1NeoPolyA plasmid (Stratagene), whereas all the
other expression plasmids were cotransfected with the
hygromycin-selectable pGEMHyg plasmid (50). Clones were
selected and maintained in the presence of 1 µg of tetracycline per
ml. Independent single clones were examined for tetracycline-dependent
expression by Western blot analysis (43) with the anti-HA1
epitope monoclonal antibody 12CA5 (Boehringer) at 2 µg/ml. Bound
antibody was visualized with a peroxidase-conjugated goat anti-mouse
antibody (Jackson) and chemiluminescence reagent (New England Nuclear)
used as specified by the manufacturer.
Cell growth, DNA content analysis, and apoptosis.
For
induction of tetracycline-controlled gene expression, cells were washed
four times with 10 ml of phosphate-buffered saline and seeded at
105 cells/ml in complete medium containing the desired
tetracycline concentration. Cell proliferation was measured by a
nonradioactive cell proliferation assay (Promega) as specified by the
manufacturer. The DNA content and cell cycle distribution were
evaluated by flow cytometric analysis of propidium iodide-stained
nuclei as described previously (37). Fragmented DNA was
isolated as previously described (32) and separated on 1%
agarose gels containing ethidium bromide.
Differentiation of U937 cells.
Induced DEK-CAN58 cells were
cultured for 5 days in the presence of 1 ng of transforming growth
factor 1 (TGF1) (Promega) per ml and 250 ng of 1,25-dihydroxy
vitamin D3 (Biomol) per ml. The percentages of monocyte
surface antigen-positive cells were evaluated by fluorescence-activated
cell sorter (FACS) analysis 5 days after induction of differentiation.
The following mouse monoclonal antibodies were used: anti-CD11a,
anti-CD11b (MO1), anti-CD14 (MY4), anti-CD15, and anti-CD18.
Indirect immunofluorescence.
Cytospins of U937 cells were
fixed, permeabilized, and immunostained as described previously
(12). Primary antibodies were diluted 1:400 for
anti-C-terminal CAN (CNC [12]), to 2 µg/ml for
12CA5, to 2 µg/ml for MAb3E9 (7), and 1:90 for
affinity-purified anti-hCRM1 (14). Bound primary antibodies
were visualized with goat secondary antibodies conjugated to
fluorescein isothiocyanate (FITC; Sigma) or Texas red (U.S.
Biochemicals). Images were obtained by confocal laser-scanning
microscopy on a Bio-Rad MRC1000 microscope with a 60× oil objective.
Transport assays.
In vitro nuclear protein import assays
were performed essentially as described previously (3), with
MDBK lysate instead of reticulocyte lysate as a source of essential
cytosolic factors. Import was allowed to proceed for 20 min at 27°C,
with parallel reactions on ice as controls. Immediately after import,
the cells were washed and fixed in 3% formaldehyde for 15 min on ice.
The mean fluorescence of the accumulated APC-NLS substrate in these cells was determined by FACS analysis. To determine the intracellular distribution of polyadenylated RNA, we hybridized cells on sterile cytospins with an oligo(dT)50 probe directly coupled to
FITC as described previously (49). Images were obtained by
confocal laser-scanning microscopy on a Bio-Rad MRC1000 microscope with a 60× oil objective.
Electron microscopy.
CAN7 cells were induced for 60 h
and then fixed for 2 h in 0.1 M phosphate buffer (pH 7.4)
containing 2% glutaraldehyde. The cells were then postfixed in 1%
osmium tetroxide for 1 h, dehydrated, and embedded in Spurr
low-viscosity resin (Electron Microscopy Sciences). Thin sections were
cut, stained with uranyl acetate and lead, and examined with a JEOL
JEM-1200EX II electron microscope.
| |
RESULTS |
CAN overexpression induces G0 arrest and
apoptosis.
To study the effects of CAN and DEK-CAN on the growth,
survival, and differentiation of myeloid cells, we introduced inducible CAN and DEK-CAN genes into the human myeloid
precursor U937 cells. First, we generated the cell line U937T, which
expressed tetVP16 in a tetracycline-dependent manner (24, 45,
51) but maintained normal growth characteristics. Subsequently,
this clone was stably transfected with expression constructs containing
HA1-epitope tagged CAN (ttCAN) or ttDEK-CAN cDNAs under the control of
the tetVP16-responsive promoter. Each cell line used for this study was
selected from a number of clonal lines based on the relative expression
level of the transfected gene after tetracycline withdrawal.
A clone that expressed relatively high levels of ttCAN upon withdrawal
of tetracycline, CAN7, was selected to study the effects on cell growth
of overexpression of different amounts of CAN by varying the
concentrations of tetracycline in the culture medium. ttCAN was
detected by immunofluorescence staining, with the anti-HA1 monoclonal
antibody, as early as 10 h after induction and reached maximal
levels after ~24 h (data not shown). Western blot analysis showed
that low concentrations of tetracycline in the culture medium (1 and 2 ng/ml) allowed the expression of intermediate levels of ttCAN whereas
complete absence of tetracycline caused greater amounts of ttCAN to
accumulate (Fig. 1A). In the presence of
3 ng of tetracycline per ml, ttCAN was barely detectable on Western
blots, whereas no protein was detected in lysates from cells cultured
at higher tetracycline concentrations. The growth curves of induced and
uninduced CAN7 cells showed that growth inhibition was directly
proportional to the amount of expressed ttCAN and inversely
proportional to the tetracycline concentration in the medium (Fig. 1B).
Like the parental U937T cells, the uninduced CAN7 cells continued to
grow with a doubling time of about 24 h, whereas cells expressing
the highest levels of ttCAN (0 and 1 ng of tetracycline/ml) stopped
growing and their numbers were reduced after 3 days. Under conditions
that gave expression of intermediate levels of ttCAN protein (2 ng of
tetracycline per ml), we observed some increase in cell number, albeit
at a significantly lower rate than that without induction.
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FIG. 1.
CAN-overexpressing cells are growth inhibited. (A)
Inducible expression of HA1-tagged CAN in CAN7 cells grown for 24 h in the presence of 1,000, 5, 3, 2, 1, and 0 ng of tetracycline per
ml, as indicated above the lanes, was assayed by Western blot analysis
of the sodium dodecyl sulfate (SDS)-6% polyacrylamide gel. Induced
parental U937T cells serve as a negative control. Each lane contains
lysate from 5 × 105 cells. The blot was probed with
the anti-HA1 monoclonal antibody 12CA5. The arrow indicates ttCAN
protein. Lysates of cells expressing large amounts of ttCAN protein
show specific truncated products, which do not seem to affect the
results. The sizes of molecular mass standards, run in an adjacent
lane, are indicated on the left in kilodaltons. (B) Growth curves of
induced CAN7 cells ( ) and U937T control cells ( ). Cultures were
maintained in medium containing the indicated tetracycline
concentrations, and viability was measured daily by a nonradioactive
proliferation assay. The relative density was calculated as a
percentage of the density of uninduced cells on day 4. Mean values of
triplicate determinations are plotted; the standard deviations were
below 10%. This experiment is one of three that all gave similar
results. (C) The cell cycle phase distribution of induced CAN7 cells
(upper panel) and U937T cells (lower panel) at 1, 2, 3, and 4 days
after tetracycline withdrawal was calculated from flow cytometric
measurements of the DNA content. (D) Flow cytometry profiles showing
DNA fluorescence of propidium iodide-stained CAN7 cell nuclei at 1, 2, 3, and 4 days after withdrawal of tetracycline. This is a
representative experiment of three, all of which gave similar
results.
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To examine if fully induced CAN7 cells arrested at a particular phase
of the cell cycle, we studied the DNA content of the
overexpressing
cells by FACS analysis of propidium iodide-stained
nuclei (Fig.
1C and
D). After 2 days of CAN induction, the percentage
of cells in S phase
was reduced from 50 to 23% and the percentage
of cells in
G
2/M was reduced from 13 to 4%. The percentage of
cells
with a diploid DNA content was increased from 35 to 67%,
suggesting
that the cells had arrested at G
1/G
0. At this
time
point, very few cells were scored with a sub-G
1 DNA
content (see
below). By day 3, however, 18% of the cells showed a
sub-G
1 DNA
content, and this fraction increased to 62% by
day 4 after tetracycline
withdrawal (Fig.
1C, upper panel). The
increase in the percentage
of hypodiploid nuclei in
ttCAN-overexpressing cells indicated
that the cells became apoptotic
(
37). Moreover, the cells displayed
morphologic features of
apoptosis, including DNA fragmentation
(Fig.
2A), nuclear segmentation, and chromatin
condensation (Fig.
2B). The cells also stained positively by the
terminal deoxytransferase-mediated
dUTP-biotin nick end labeling method
(
17), which detects double-strand
DNA breaks that are
indicative of apoptosis (data not shown).
U937T cells grown in the
absence of tetracycline continued to
cycle normally. They reached
confluency by day 4, resulting in
more cells in
G
1/G
0 and fewer cells in S and
G
2/M. In contrast
to ttCAN-overexpressing cells, the
percentage of cells with a
sub-G
1 DNA content remained low,
between 2 and 4% (Fig.
1C, lower
panel).
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FIG. 2.
Overexpression of CAN induces G0 apoptosis
in U937T cells. (A) Agarose gel electrophoresis of DNA from CAN7 cells
cultured for 3 days in the absence of tetracycline (lane 3) shows
internucleosomal DNA cleavage, whereas DNA from the parent U937T cells
grown in the presence (lane 1) or absence (lane 2) of 1 µg of
tetracycline per ml remains unfragmented. PstI-digested  DNA served as a molecular weight marker (lane M). (B) Electron
micrographs showing apoptotic CAN-overexpressing CAN7 cells after 3 days of induction. Bar, 2 µm. (C) Northern blot analysis of 15 µg
of RNA isolated from the total culture (lane 1) and the FACS-sorted
diploid-cell fraction (lane 2) of CAN7 cells induced for 40 h,
compared to the total cultures of uninduced CAN7 cells (lane 3) and
induced parental U937T cells (lane 4). The amounts of c-myc
(2.4 kb; top panel), and cyclin D2 (6.0 kb; middle panel)
mRNA were compared to the levels of actin mRNA (2.0 kb; bottom
panel).
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To determine if CAN-arrested CAN7 cells were blocked in G
1
or G
0, we studied their c-
myc mRNA expression
levels. Cycling cells
express high levels of c-
myc mRNA,
whereas cells that exit the
cell cycle and arrest in G
0 do
not transcribe c-
myc (
29). CAN7
cells were
induced in the absence of tetracycline for 40 h and
RNA was
isolated from the total culture and from cells sorted
for a diploid DNA
content. RNA from total cultures of uninduced
CAN7 cells and parental
U937T cells was isolated to serve as a
control. Northern blot analysis
showed a dramatic decrease in
the amount of c-
myc mRNA in
the ttCAN-expressing cells compared
to that expressed in uninduced
cells. The low level of c-
myc mRNA
in the total culture was
derived from the fraction of cells that
remained cycling, since the
sorted diploid-cell fraction was negative
for c-
myc mRNA
(Fig.
2C, top panel). A similar, albeit smaller,
reduction was observed
in the amounts of cyclin D
2 mRNA (Fig.
2C, middle panel),
which is normally present throughout the cell
cycle (
44).
The more rapid downregulation of c-
myc could be
caused by
the very short half-life of c-
myc mRNA (
53).
These
data strongly suggest that, prior to apoptosis, the
ttCAN-overexpressing
cells exited the cell cycle and arrested in
G
0.
Bcl-xL coexpression does not prevent apoptosis.
Bcl-xL, a member of the Bcl-2 family, can protect cells
against a variety of apoptosis inducers (5, 19, 25). We
examined whether constitutive coexpression of the Bcl-xL
gene with ttCAN in CAN7 cells could inhibit CAN-induced apoptosis. A
representative stably transfected CAN7 clone, CAN7-B2, expressed high
levels of Bcl-xL protein and grew slower than the parental
CAN7 clone (Fig. 3). This could be a
direct consequence of Bcl-xL expression, since expression
of this protein has been shown to prolong the G1 phase in
U937 and other cell lines (6). Bcl-xL expression had no influence on growth inhibition or cell death after tetracycline was removed from several independent CAN7-Bcl-xL clones
(Fig. 3), indicating that Bcl-xL could not inhibit the
apoptotic cell death of induced CAN7 cells.
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FIG. 3.
Bcl-xL coexpression does not rescue
CAN-overexpressing cells. Growth curves of a representative
Bcl-xL-overexpressing CAN7 clone, CAN7-B2 ( ), compared
to CAN7 (), both grown in 1,000, 2, and 0 ng of tetracycline per ml
for 4 days. Mean relative density values of triplicate cultures are
plotted against time; the standard deviations were below 10%. This
experiment is one of three, all of which gave similar results. Inset:
Western blot of an SDS-9% polyacrylamide gel containing lysate from
5 × 105 cells per lane, probed with a mouse
monoclonal antibody to Bcl-xL. The 29-kDa doublet
represents the Bcl-xL protein. The position of the 28-kDa
molecular mass standard is indicated on the right.
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Transport defects in CAN-overexpressing cells.
We studied the
subcellular localization of CAN in induced and uninduced cells by
indirect immunofluorescence with the polyclonal CAN antiserum CNC
(12). In the parental U937T cells, endogenous CAN levels
were low and the protein localized to the nuclear envelope (Fig.
4A). During the first day after
tetracycline withdrawal, ttCAN in CAN7 cells was localized mainly to
the nuclear envelope and cytoplasmic speckles. The latter structures
could be annulate lamellae or simply aggregates of insoluble protein
(Fig. 4B). However, during the second day after induction, an
increasing percentage of cells showed nuclear localization of ttCAN,
and by the third day, 90% of the cells had ttCAN in the nucleus (Fig. 4C). The nuclear staining was diffuse, with a few strong dots. These
results demonstrate that the loss of cell viability upon ttCAN
overexpression coincides with the accumulation of ttCAN in the
nucleoplasm.
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FIG. 4.
Polyadenylated RNA export defect in CAN-overexpressing
cells. (A to C) Confocal images of endogenous CAN expression in U937T
cells (A) and overexpressed CAN in CAN7 cells 20 h (B) and 48 h (C) after induction; the cells were stained with the anti-CAN
antiserum CNC. (D) Overexpression of HA-CAN1864-2090 in U937T cells
stained with the anti-HA antibody 12CA5. (E to H) Subcellular
localization of polyadenylated RNA in control U937T cells (E) and CAN7
cells after 48 h (F) and 60 h (G) of induction and in HA-CAN
1864-2090 cells after 60 h of induction (H) analyzed by in situ
hybridization with an FITC-conjugated oligo(dT)50 probe.
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Because CAN is a nucleoporin, we asked whether the cytotoxity of its
overexpression coincided with perturbations in its NPC
transport
function. We measured the nuclear export capacity of
the
ttCAN-overexpressing cells by monitoring the localization
of
polyadenylated RNA by in situ hybridization with an
oligo(dT)
50 probe directly coupled to FITC (
4).
At 48 h after tetracycline
withdrawal, poly(A)
+ RNA in
both U937T cells (Fig.
4D) and CAN7 cells (Fig.
4E) appeared
to be
diffusely distributed throughout the nucleoplasm and cytoplasm.
In
contrast, at 56 to 60 h after ttCAN induction, only CAN7 cells
showed strong nuclear staining, indicating nuclear accumulation
of
poly(A)
+ RNA (Fig.
4F). At this time point, most cells were
still alive
and arrested in G
0. As a control, we induced
apoptosis by culturing
U937T cells for 48 h in the presence of 50 µg of the protein synthesis
inhibitor cycloheximide per ml. In this
case, apoptosis was not
preceded by mRNA accumulation in the nucleus
(data not shown),
suggesting that the block in mRNA export observed in
CAN7 cells
was specific for CAN overexpression.
The nuclear protein import capacity of induced CAN7 cells was assessed
in vitro by examining the accumulation of an NLS-linked
fluorescent
substrate, APC-NLS, in the nuclei of digitonin-permeabilized
cells
(
3). After 24 h of tetracycline withdrawal,
ttCAN-overexpressing
CAN7 cells showed an import capacity similar to
that of U937T
cells whereas CAN7 cells that had been induced for
48 h did not
appreciably import APC-NLS (data not shown). These
results indicated
that the inhibition of nuclear protein import in
CAN-overexpressing
cells coincided with the cell cycle arrest.
Therefore, we compared
the import capacity of CAN7 cells 2 days after
tetracycline withdrawal
with that of U937T cells that had reached their
maximal density
4 days after seeding. In both cultures, almost 70% of
the cells
were arrested with a diploid DNA content (Fig.
1C).
Unexpectedly,
we could not detect NLS protein import in the
density-arrested
U937T cells either, which makes it impossible to
distinguish between
cell cycle arrest and CAN overexpression as the
primary cause
of the import defect.
Inhibition of nucleocytoplasmic transport may be caused by gross
structural alterations in the NPC or nuclear envelope or
by functional
perturbation. Thin-section electron microscopy did
not reveal
structural perturbations of the NPC or nuclear envelope
in
CAN-overexpressing CAN7 cells after 3 days of induction (data
not
shown). Therefore, we assessed whether the toxicity of excess
CAN
resulted from functional inactivation of CAN-interacting proteins.
hCRM1, a protein that coimmunoprecipitates with CAN, shuttles
between
the nucleus and the cytoplasm and functions as an export
receptor for
leucine-rich nuclear export sequences (
14,
46).
We used
immunopurified polyclonal antiserum to detect hCRM1 (
14)
in
combination with a monoclonal antibody, 12CA5, to the HA1 epitope
to
detect ttCAN in double-immunostaining experiments. Strikingly,
we found
that endogenous hCRM1 colocalized with overexpressed
CAN in the nuclear
envelopes, cytoplasm, and nuclei of CAN7 cells
(Fig.
5A and
B). In cells that expressed CAN in both
cytoplasmic
and nuclear speckles, hCRM1 preferentially colocalized with
the
nuclear structures (Fig.
5A and B). The accumulation of ttCAN
in
the nuclei of CAN7 cells after 3 days resulted in colocalization
of
hCRM1 in the nucleus and depletion of this factor from the
nuclear
envelope. We then studied the localization of the transport
factor
p97/importin
, which binds to CAN and other FG-repeat
containing
nucleoporins in vitro and localizes to the NPC and
cytoplasm (
7,
23,
39). We immunostained induced CAN7 cells
with a monoclonal
antibody to p97 (MAb3E9 7) and anti-CNC and
found that p97 colocalized
with ectopically expressed CAN protein
in the nuclear envelope and the
cytoplasmic speckles. Two days
after ttCAN induction, colocalization
also occurred in the nucleus,
which resulted in depletion of p97 from
the nuclear envelope (Fig.
6A and B).
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FIG. 5.
Colocalization of full-length CAN and CAN mutants with
hCRM1. The subcellular distribution of hCRM1 and CAN proteins after 2 days of induction is shown. (A, C, E, G, I, and K) Confocal microscopy
showing immunodetection of induced CAN and mutant proteins by an
anti-HA1 monoclonal antibody followed by a goat anti-mouse Texas
red-conjugated secondary antibody. (B, D, F, H, J, and L) Distribution
of endogenous hCRM1 in the same cells stained with the anti-hCRM1
antiserum and a goat anti-rabbit FITC-linked antibody. hCRM1
colocalized with full-length CAN (A and B [cells indicated by
arrows]), with the C-terminal FG repeat regions of CAN, CAN 1864-2090
(C and D) and CAN (1140-1340, 1864-1912, 1984-2090) (E and F), and
with DEK-CAN (I and J). In contrast, hCRM1 did not colocalize with the
more N-terminally located FG repeat region of CAN, represented by CAN
1558-1840 (G and H); it showed normal distribution in the nuclear
membrane and the nucleoplasm, similar to that of induced U937T cells (K
and L).
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FIG. 6.
Double immunostaining of CAN and mutants with
p97/importin . The subcellular distribution of p97 and CAN proteins
after 2 days of induction is shown. (A, C, E, and G) Confocal images of
indirect immunofluorescence with the anti-CAN polyclonal antiserum
anti-CNC, detected with a Texas red-conjugated goat anti-rabbit
antibody. (B, D, F, and H) Confocal images of the same cells
immunostained with an antibody to p97 (MAb3E9), detected with an
FITC-conjugated goat anti-mouse antibody. Endogenous p97 colocalized
with overexpressed CAN in the nuclear membrane and in the cytoplasmic
and nucleoplasmic speckles (A and B). In cells overexpressing CAN
816-2090, only the nuclear membrane and the cytoplasmic speckles
showed colocalization of the CAN mutant with p97 (C and D). Cells
overexpressing CAN 1140-2090 showed normal p97 localization in the
nuclear membrane (E and F), comparable to cells expressing endogenous
levels of CAN (G and H).
|
|
Expression of DEK-CAN and CAN mutants in U937 cells.
To map
the region of CAN that mediated cycle arrest and apoptosis, we studied
the response of U937T cells to induced expression of CAN mutants and
the leukemia-specific DEK-CAN fusion protein (Fig.
7). Clones were selected for high levels
of expression after induction; however, some mutants, including
DEK-CAN, did not reach the expression levels of ttCAN (Fig.
8A). Most of the mutants described in
this study were analyzed previously for subcellular localization and
coimmunoprecipitating proteins in transient-transfection studies
(13). The N terminus of CAN (CAN 1-1058) and a shorter central region (CAN 589-1058) both associate with the NPC and bind
NUP88. Expression of these mutants did not affect cell growth or
viability (Fig. 8B). However, the expression of C-terminal regions of
CAN, such as CAN 816-2090, which localizes to the NPC, cytoplasm, and
nucleus, and the nuclear mutant CAN 1140-2090, inhibited cell growth
(Fig. 8B). This effect could be attributed to overexpression of the
most C-terminal FG repeat-containing region (CAN 1864-2090), which was
sufficient for the lethal phenotype (Fig. 8B). Importantly, this part
of CAN harbors the hCRM1-binding domain. DNA histogram analysis (Fig.
8C and D) of induced CAN 1864-2090 cells showed that 2 days after
tetracycline withdrawal, the percentage of cells in S phase was reduced
from 50 to 25% and the percentage of cells in
G0/G1 had increased from 36 to 58%. The
percentage of cells with a sub-G1 DNA content was 27% after 3 days of induction and 40% after 4 days (Fig. 8C). This demonstrates that overexpression of the hCRM1-binding region is sufficient to cause cycle arrest and cell death. Although the entire
cell population underwent apoptosis, the onset was slower than when the
full-length CAN was overexpressed, possibly because of subtle
differences in the expression levels between the two clones. CAN
deletion mutants that lack part of the region necessary for hCRM1
coimmunoprecipitation [CAN (1140-1340, 1864-1912, 1984-2090) and
CAN 1864-2017] still localized to the nucleus and exhibited the
lethal phenotype, albeit at considerably higher expression levels (Fig.
8A and B). The FG repeat-containing region just N-terminal of the
hCRM1-binding region (CAN 1558-1840) localized to the cytoplasm and
did not affect cell growth (Fig. 8B). The most highly inducible DEK-CAN
clone, DEK-CAN58, expressed only about 25% of the amount of CAN
protein produced in CAN7, as estimated by Western blot analysis (Fig.
8A). This clone was slightly growth inhibited but did not die of
apoptosis (Fig. 8B).
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FIG. 7.
Overview of CAN deletion mutants. Black bars represent
CAN and CAN mutant proteins; numbers on the left represent amino acid
boundaries. Predicted structural motifs are represented as follows:
vertical lines, FG repeats; diamonds, FXF repeats; LZ, coiled-coil 1 and adjacent leucine zipper; AH, coiled-coil 2. Horizontal stripes
indicate an acidic region in the DEK sequence (white bar).
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FIG. 8.
The C terminus of CAN is sufficient to inhibit cell
growth. (A) Western blot analysis of SDS-6% polyacrylamide (left
panel) and SDS-10% polyacrylamide (right panel) gels with lysates
from 5 × 105 induced cells expressing the indicated
CAN mutants and DEK-CAN. CAN (1140-1340, 1864-1912, 1984-2090) is
abbreviated to CAN 1864 1912+1984290. Blots were probed as described
for Fig. 1A. (B) Growth curves of cells overexpressing selected CAN
mutants and DEK-CAN grown in the absence of tetracycline for 4 days.
Data shown are the mean values of triplicate cultures from one
experiment of at least three independent experiments that gave similar
results. (C) Cell cycle phase distribution of induced CAN 1864-2090
cells. (D) Quantitation of the DNA content by flow cytometric analysis
in CAN 1864-2090 cells, overexpressing the hCRM1-binding domain, on
days 1, 2, 3, and 4 after withdrawal of tetracycline.
|
|
Double-immunofluorescence staining studies of CAN mutants (Fig.
5, left
panel) with hCRM1 (Fig.
5, right panel) showed that
endogenous hCRM1
colocalized with CAN 816-2090 in the cytoplasm
and in the nucleus
(data not shown). The endogenous hCRM1 also
colocalized with the
hCRM1-binding domain (CAN 1864-2090) in the
nucleus (Fig.
5C and D).
The nuclear mutants CAN (1140-1340, 1864-1912,
1984-2090) (Fig.
5E
and F) and CAN 1864-2017 (data not shown),
which both lack part of the
hCRM1-binding domain that is required
to coimmunoprecipitate hCRM1
(
13), still caused redistribution
of hCRM1 to the nucleus.
In contrast, CAN 1558-1840, which also
does not coprecipitate hCRM1
(
13), did not colocalize with hCRM1
(Fig.
5G and H) or
influence cell growth. Although DEK-CAN levels
were not high enough to
induce cell death, the staining patterns
of DEK-CAN and hCRM1 were
overlapping in highly expressing cells
(Fig.
5I and J). Taken together,
all (partly) nuclear CAN mutants
colocalize with hCRM1, suggesting that
the region of CAN needed
for in vivo interaction with hCRM1 is smaller
than the hCRM1-binding
domain identified by coimmunoprecipitation. High
expression levels
of these mutants resulted in sequestration of hCRM1
in the nucleus
and depletion of hCRM1 from the nuclear envelope, which
coincided
with growth inhibition and cell death.
We studied the distribution of endogenous p97 (Fig.
6, right panel) in
cells expressing CAN mutants (Fig.
6, left panel) and
found
colocalization of p97 with CAN 816-2090 in the nuclear envelope
and
the cytoplasmic speckles. Cells expressing this CAN mutant
in nuclear
speckles showed a normal p97 distribution (Fig.
6C
and D). Expression
of DEK-CAN (data not shown) or CAN 1040-2090
(Fig.
6E and F) did not
affect p97 localization, indicating that
sequences in the central
region of CAN mediate this effect.
Poly(A)
+ RNA in clones expressing DEK-CAN (data not shown)
or the C terminus of CAN (Fig.
4H) was diffusely distributed in
the
nucleoplasm and cytoplasm, similar to U937T control cells
(see Fig.
4D), indicating that there was no defect in mRNA export
in these cells.
Thus, it is unlikely that hCRM1 is involved in
the export of mRNA.
Digitonin-permeabilized cells overexpressing
the hCRM1-binding domain
were severely inhibited in APC-NLS import,
comparable to
CAN-overexpressing cells and density-arrested U937T
cells (data not
shown). Therefore, a possible additional effect
on import of hCRM1
sequestering in the nucleus could not be determined.
DEK-CAN does not inhibit differentiation of U937 cells.
To
study the effect of DEK-CAN on myeloid maturation, we induced clone
DEK-CAN58 to differentiate by using a combination of transforming
growth factor 1 (TGF1) and 1,25-dihydroxy vitamin D3
(D3) (48) after 3 days of withdrawal from tetracycline.
Surprisingly, the cells died rapidly (Fig.
9A). Immunofluorescence staining showed
elevated levels of DEK-CAN in these cells compared to those in
undifferentiated cells (data not shown). We consistently found this
effect, even when differentiation was induced by other chemicals, such
as dimethyl sulfoxide and phorbol-12-myristate-13-acetate. We therefore
tried partially releasing the DEK-CAN58 cells for 3 days in the
presence of 5 and 10 ng of tetracycline per ml prior to inducing
differentiation. Both partially induced and uninduced cells ceased to
proliferate upon exposure to TGF1 and D3 (Fig. 9A). Compared to
uninduced cells (in 1,000 ng of tetracycline per ml), viability was not
affected in cells cultured with differentiation agents in 10 ng of
tetracycline per ml, whereas about 50% of the cells grown in 5 ng of
tetracycline per ml died. The remaining cells in these cultures
expressed high levels of DEK-CAN.
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FIG. 9.
Differentiation antigen expression in U937T cells
expressing DEK-CAN. (A) Growth curves of DEK-CAN58 cells cultured in
medium containing 1,000 ( ), 10 ( ), 5 ( ), or 0 () ng of
tetracycline per ml, seeded at 105 cells/ml in the presence
(differentiated) or absence (undifferentiated) of 1 ng of TGF1 per
ml and 250 ng of D3 per ml. The cells were cultured in normal medium
containing the indicated tetracycline concentrations for 3 days prior
to the induction of differentiation. (B) DEK-CAN58 cells cultured in
1,000 ng of tetracycline per ml (no DEK-CAN expression) and 5 ng of
tetracycline per ml (partial DEK-CAN induction) were differentiated for
5 days in medium containing TGF1 and D3 (differentiated; black
bars). Undifferentiated cells cultured in 1,000 and 5 ng of
tetracycline per ml are also shown (undifferentiated; gray bars).
Expression of the indicated differentation antigens (CD11a, CD11b,
CD14, CD15, and CD18) was evaluated by cytofluorimetry with specific
monoclonal antibodies. Results are expressed as percentages of
antigen-positive cells.
|
|
DEK-CAN58 cells grown in 5 and 1,000 ng of tetracycline per ml were
induced to differentiate by a 5-day exposure to TGF
1
and D3. As a
measure of induction of differentiation, we monitored
the increased
expression of the cell surface antigens CD11a, CD11b,
CD14, and CD18.
The enhanced expression of these markers indicated
that
DEK-CAN-expressing DEK-CAN58 cells (Fig.
9B, right panel)
differentiated normally to mature monocytes in a manner
indistinguishable
from that of tetracycline-repressed DEK-CAN58 cells
(Fig.
9B,
left panel). Moreover, Giemsa staining of the two
differentiated
cell populations showed the same morphology (data not
shown),
confirming that DEK-CAN expression did not inhibit the myeloid
differentiation of U937 cells.
| |
DISCUSSION |
We examined the biologic properties of the CAN protein and the
leukemia-specific DEK-CAN fusion protein by overexpressing these
proteins in U937 myeloid precursor cells. We found that ectopic
expression of CAN caused a cell cycle arrest in G0 followed by a block in mRNA export and apoptotic cell death. Overexpression of
the most C-terminal FG repeat-containing region of CAN, which binds
hCRM1, was sufficient to reproduce most of these effects: it induced
growth arrest and apoptosis. Thus, overexpression of CAN and the
hCRM1-binding domain of CAN interfered with some of the normal
functions of CAN in a dominant-negative way. Considering the large
number of importin family members, the effects of the C-terminal
fragment of CAN on apoptosis could conceivably be related to
interfering with another transport factor besides hCRM1. However, in
immunoprecipitation experiments, only hCRM1 coprecipitates with the
C-terminal FG repeat (13) and is therefore most likely to be
responsible. Moderate DEK-CAN expression slightly inhibited cell growth
and did not interfere with the differentiation of U937 cells to mature
monocytes. Because U937 cells represent an intermediate stage of
monocytic development (26), it is possible that their
terminal differentiation is not affected because DEK-CAN may inhibit
the differentiation only of earlier myeloid precursors. Myeloid cells
from patients with t(6;9) acute myeloid leukemia are partially
inhibited in their differentiation pathways but are not totally
blocked. Therefore, it is also possible that DEK-CAN has no effect on
differentiation but affects the proliferation of early precursor cells
in these patients.
CAN overexpression induces cell cycle arrest and apoptosis.
CAN-overexpressing cells and, to a lesser extent, cells overexpressing
the hCRM1-interaction domain of CAN accumulated with a diploid DNA
content. Arrested CAN-overexpressing cells no longer express
c-myc, a proto-oncogene that is continuously expressed in
proliferating cells but is downregulated when cells exit the cell cycle
(15, 53). U937T cells that were mostly arrested in
G0/G1 after reaching their maximal density did
not import detectable levels of import substrate. Therefore, additional
effects of the overexpression of CAN and the hCRM1-binding domain on
nuclear protein import could not be measured. It is possible that CAN overexpression interferes with the nuclear transport of factors critical to cell growth or survival. Alternatively, proper
stoichiometry of the components that make up the NPC could be necessary
for the formation of new NPCs, a process that is presumably essential for growth. CAN depletion leads to cell cycle arrest in G2
(49), whereas overexpression of CAN arrested cells in
G0, suggesting that CAN is essential to proper cell cycle
progression. Furthermore, CAN-depleted cells still have hCRM1 in their
nuclear envelopes (14), whereas the nuclear envelopes of
CAN-overexpressing cells are depleted of hCRM1 (see below). It will be
interesting to see how the transport function of hCRM1 could be linked
to cell cycle progression.
Cells overexpressing full-length CAN or CAN mutants containing the
hCRM1-binding domain die after 72 to 96 h of induction
and show
morphologic features of apoptosis. Coexpression of the
potent survival
factor Bcl-x
L did not protect CAN-overexpressing
cells from
apoptosis, suggesting that a non-Bcl-x
L-controlled
pathway
is activated.
hCRM1 function is essential.
hCRM1 normally localizes to the
nucleus and the NPC and is regularly released from the NPC into the
nucleoplasm (14). CAN mutants containing the hCRM1-binding
region were, at least in part, nuclear and caused nuclear accumulation
of hCRM1. Overexpression of these mutants at levels that were lethal
resulted in a complete depletion of hCRM1 from the nuclear envelope.
This result suggests that the excess of the nuclear hCRM1-binding
domain of CAN competes with NPC-associated CAN for binding to hCRM1 and
inhibits a function of hCRM1 that is essential for cell growth or
survival. In the light of recent findings, this essential function of
hCRM1 most probably is the export of NES-containing (ribonuclear)
proteins, among which are UsnRNPs (14a, 46). Inhibition
of UsnRNP export prevents their cytoplasmic modification and may
eventually affect pre-mRNA splicing, which could be an effective
inducer of apoptosis. It would be interesting to see if progressive
inhibition of other essential nuclear transport factors also causes
apoptosis or if this is specific to hCRM1. Why do cells arrest in
G0 if hCRM1 is a general nuclear export factor? Besides
possible defects in the transport of specific NES-containing proteins,
it is conceivable that inhibition of splicing could preferentially
affect the production of short-lived mRNAs, encoding proteins with a
high turnover rate, such as c-MYC, that directly affect the ability of
the cell to enter the G1 phase.
The localization of overexpressed full-length CAN changed from mainly
nuclear membrane and cytoplasm after the first day of
induction to
mainly nuclear during the second and third days of
expression. HeLa
cells, transiently transfected to highly overexpress
CAN, showed a
similar nuclear localization in 5 to 10% of the
cells (
12).
It may be that overexpressed CAN spills over into
the cytoplasm and
nucleus when all of the NPC-binding sites are
saturated. Transport of
CAN into the nucleus could be mediated
by its association with hCRM1
(
13). The colocalization of CAN
and hCRM1 in cytoplasmic
structures suggests that complex formation
has already occurred in the
cytoplasm. Since hCRM1 has a half-life
of approximately 24 h
(
14), it is unlikely that the hCRM1 observed
in the
cytoplasm of CAN-overexpressing cells is only newly synthesized
hCRM1.
Instead, these data suggest that hCRM1 travels from the
nucleus to the
cytoplasm, in addition to its release from the
NPC into the
nucleoplasm. This would be in agreement with the
shuttling function of
CRM1 in yeast (
46). In addition, the movement
of hCRM1
between the nucleus and the cytoplasm was confirmed by
microinjection
studies in
Xenopus oocytes (
14).
The region of CAN required for its colocalization with hCRM1 in the
nucleus was smaller than the hCRM1-binding domain identified
by
coimmunoprecipitation (
13), suggesting that the in vivo
interaction
of mutants that lack part of the binding domain is too weak
to
be detected by immunoprecipitation. This finding is consistent
with
the idea that nuclear localization of the C-terminal repeat
region of
CAN, which does not contain a known NLS, is mediated
by hCRM1
(
13).
CAN overexpression induces defects in p97 localization and mRNA
export.
Overexpressed CAN colocalized with the nuclear import
factor p97/importin , initially in the nuclear membrane and
cytoplasmic structures and subsequently in nuclear structures. p97
binds to CAN in vitro (39), but this interaction is not
strong enough to mediate coimmunoprecipitation with CAN from cell
lysates of CAN-overexpressing cells (14). Based on binding
studies of NUP98 mutants and other nucleoporins, the FG repeat regions
are thought to harbor the p97-binding domain (36, 40). Our
results with CAN 1558-1840 and CAN 1864-2090 show that p97 does not
bind to the overexpressed C-terminal CAN repeat regions alone. Instead, p97 colocalizes with full-length CAN and partly with CAN 817-2090, both of which bind to the NPC and form structures of unknown
composition upon overexpression. It is possible that p97 also
associates in vivo with the central region of CAN, either directly or
via another protein, and that binding of p97 to CAN requires both
additive interactions.
Only cells overexpressing full-length CAN demonstrated a defect in
polyadenylated RNA export after 55 to 60 h of induction.
These
were also the only cells to sequester p97 in their nuclei.
These
observations may be directly linked because depletion of
p97 from the
cytoplasm may lead to a block in importin
import
into the nucleus,
thereby inhibiting its function in the export
of capped RNAs
(
20). Studies with yeast cells overexpressing
the
nucleoporin gene
NUP116 also suggest that p97 plays a role
in mRNA export. Overexpression of the GLFG repeat region of Nup116p
severely inhibits cell growth and blocks polyadenylated-RNA export
(
27). This region interacts with Kap95p, an essential yeast
homolog of the vertebrate import factor p97, suggesting that
sequestering
of this factor is at least partly responsible for the
phenotype
(
27).
In summary, our results indicate that the cytotoxicity of CAN
overexpression may be caused by depleting hCRM1 from the nuclear
envelope and confining it to the nucleus, thereby inhibiting the
export
of NES-containing substrates. Furthermore, only full-length
CAN, which
contains the central protein-protein interaction domain
in addition to
the C-terminal FG repeats, colocalizes with p97
in the nucleus and
causes nuclear accumulation of polyadenylated
RNA.
The mechanism by which DEK-CAN contributes to leukemogenesis remains
unknown. DEK is a sequence-specific DNA-binding protein,
and
DEK-binding sites were recently identified in the regulatory
regions of
several early myeloid genes (
16). DEK-CAN could exhibit
altered transcriptional regulation compared with DEK, due to the
presence of CAN sequences or proteins that associate with this
portion
of CAN, such as hCRM1. Alternatively, the redistribution
of hCRM1
towards the nucleoplasm by DEK-CAN could interfere with
the transport
function of CAN and hCRM1. DEK-CAN is expressed
at such a low level in
leukemic cells that a total depletion of
hCRM1 from the NPC is not to
be expected but a shift in the balance
of nuclear hCRM1 may have an
effect on hematopoietic cell growth
or differentiation.
| |
ACKNOWLEDGMENTS |
We are grateful to Dario Vignali for pUHD/TetVP16Puro,
Gabriel Nuñez for the spleen focus-forming virus
Bcl-xL plasmid, John Cleveland for a c-myc
probe, Charles Sherr for a cyclin D2 probe, Stephen Adam
for monoclonal antibody MAb318, and Maarten Fornerod for
affinity-purified hCRM1 antibodies. We thank Sharon Frase and Andrea
Elberger for use of the Confocal Laser Scanning Facility, UT Memphis
(funded by PHS grant CLSM 1S10RR08385), Donna Davis and Gopal Murti for
electron microscopic studies, Richard Ashmun for FACS analyses, Sjozef
van Baal for help with the figures, Charlette Hill for secretarial
assistance, and Sue Vallance for scientific editing.
These studies were supported in part by Cancer Center CORE grant
CA-21765 and by the Associated Lebanese Syrian American Charities (ALSAC) of St. Jude Children's Research Hospital.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Genetics, St. Jude Children's Research Hospital, 332 N. Lauderdale,
Memphis, TN 38105. Phone: (901) 495-2279. Fax: (901) 526-2907. E-mail: gerard.grosveld{at}stjude.org.
| |
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Mol Cell Biol, March 1998, p. 1236-1247, Vol. 18, No. 3
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
