From the Departments of Medicine and
¶ Physiology and § School of Biological Sciences,
University of Liverpool, Liverpool L69 3GA, United Kingdom
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ABSTRACT |
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The Gal Many lectins affect the proliferation of cells to which they bind;
classic examples include concanavalin A and phytohaemagglutinin as well
as more surprising mitogens such as solubilized hepatocyte asialoglycoprotein receptor (1). Some lectins affect cell proliferation by cross-linking of cell surface glycoconjugates probably without any
need for internalization (2, 3), whereas ricin is internalized and
translocated to the cytosol, where its associated toxin exerts its
cytotoxic effects (4). In many cases, however, the mechanism for the
effects of lectins on proliferation is unclear.
Increased cell surface expression of the oncofetal carbohydrate
antigen, Thomsen-Friedenreich
(TF,1 Gal Cell Proliferation, RNA, and Protein Synthesis
The HT29 cell line, established from an adenocarcinoma from a
44-year-old female Caucasian, was obtained from the European Cell
Culture Collection (Public Health Laboratory Service, Porton Down
Wiltshire, UK) and cultured as described previously (14).
Subconfluent HT29 cells cultured in Dulbecco's modified Eagle's
medium (DMEM) containing 5% fetal calf serum (FCS) (v/v) in 24-well
plates were washed twice with PBS, and serum-free DMEM containing 250 µg/ml bovine serum albumin (BSA) was added. After incubation at
37 °C for 24 h, ABL (Sigma), agarose immobilized-ABL (EY
Laboratories Inc., San Mateo, CA), 10-nm colloidal
gold-conjugated ABL (EY Laboratories) or FITC-conjugated ABL (EY
Laboratories) was added for 24 h before a 1-h pulse with 0.8 µCi/well [methyl-3H]thymidine. The cell-associated
radioactivity was determined as described previously (15).
To assess the effect of ABL on DNA, RNA, and protein synthesis in
subconfluent cells, the culture medium was replaced by serum-free DMEM
containing 250 µg/ml BSA, and 20 µg/ml ABL was introduced. The
cells were pulsed with 1 µCi/ml [methyl-3H]thymidine,
[3H]uridine, or [3H]leucine for 1 h at
the time indicated in the figure legends, and the radioactivity
associated with macromolecules was determined as described previously
(15).
Internalization Assessed by Electron Microscopy
Gold-ABL--
HT29 cells were cultured in DMEM containing 5%
FCS (v/v) in 24-well plates until 80% confluent. The culture medium
was replaced by serum-free DMEM containing 250 µg/ml BSA and 5 µg/ml 10-nm colloidal gold-conjugated ABL. In control wells, the
medium was replaced with either serum-free DMEM containing 250 µg/ml
BSA or 5 µg/ml 10-nm colloidal gold (Sigma). The cells were incubated at 37 °C for 1, 2, 4, 6, 8, 12, or 24 h. After three washes
with filtered PBS, the cells were released from the plates by
trypsinization. Cells were pelleted by centrifugation at 1000 × g for 5 min and then fixed in 2.5% glutaraldehyde (v/v)
(electron microscopy grade, Agar Scientific Ltd., Stansted, UK) in
Sorensen's buffer, pH 7.4. The samples were processed for conventional
electron microscopy using a standard 3-day processing protocol, stained
with uranyl acetate and Reynold's lead citrate, and viewed on a
Philips CM-10 electron microscope.
FITC-ABL--
HT29 cells were cultured as above in the presence
of 0 to 40 µg/ml FITC-ABL for 1 or 24 h. The cells were
released, washed, and pelleted as above before fixation in 2%
paraformaldehyde and 0.1% glutaraldehyde in Sorensen's buffer. Cell
pellets were dehydrated through an ethanol series before embedding in
LR White (London Resin Co., UK). Sections were incubated with rabbit
anti-fluorescein antibody (DAKO Ltd., Cambridge, UK) followed by gold
(10 nm)-conjugated goat anti-rabbit antibody (Sigma). On some sections
the gold labeling was silver-enhanced (British Biocell International,
UK) before staining with uranyl acetate and Reynold's lead citrate.
Control sections included untreated cells and omission of primary
antifluorescein antibody.
Intracellular Distribution of FITC-ABL and Tetramethylrhodamine
Isothiocyanate (TRITC)-Dextran Analyzed by Confocal Microscopy
Subconfluent HT29 cells cultured in glass-bottomed dishes
(MatTek Corp. Ashland, MA) were incubated at 37 °C with 20 µg/ml FITC-ABL in serum-free DMEM containing 250 µg/ml BSA. After 1 h,
unbound lectin was removed by three washes with PBS, and the cells were
cultured in fresh serum-free DMEM containing 250 µg/ml BSA at
37 °C in a CO2 chamber attached to a LSM510
laser-scanning confocal microscope (Carl Zeiss, Jena, Germany). The
FITC distribution was then monitored every h for 24 h.
In dual-label experiments, the cells were incubated for 30 min with
0.15 µg/ml TRITC-dextran (155 kDa, Sigma) followed by the addition of
2 µg/ml FITC-ABL. The subcellular distributions of FITC and TRITC
were monitored simultaneously using the 488-nm and 543-nm laser lines
every h for 10 h.
Effect of ABL on Nuclear Import of Heat Shock Protein
HT29 cells were cultured on glass coverslips in 24-well plates
in DMEM containing 5% FCS (v/v) at 37 °C for 5 days. The cells were
washed once with PBS and cultured in DMEM containing 1% FCS (v/v) for
a further 24 h at 37 °C. ABL (0-80 µg/ml) or PNA (40 µg/ml) was added, the same volume of diluent (PBS) was added to control cells, and the cells were then cultured at 37 °C for 6 h before being placed into a 43 °C incubator (5%
CO2-95% air) for 1 or 3 h of heat stress. The time
course of the ABL effect was also assessed by varying the length of
incubation of HT29 cells with 80 µg/ml ABL from 0-24 h before heat
shock. The cells were then washed 3 times with ice-cold PBS and fixed
overnight with 3.5% paraformaldehyde (w/v) and 0.1% Triton-100 (v/v)
in PBS at 4 °C (18). The fixed cells were washed 3 times with
ice-cold PBS and then washed with PBS containing 1% BSA (w/v) and 1%
Triton X-100 (v/v) for 10 min at room temperature. Nonspecific binding was blocked by incubation with 5% normal rabbit serum (v/v) for 1 h at room temperature. Mouse IgG1 monoclonal antibody, which recognizes
Hsp73 (Sigma) (1:50 dilution with 1% BSA (w/v) in PBS) or mouse IgG1
monoclonal antibody to inducible Hsp72 (Amersham Pharmacia Biotech)
(1:200 dilution with 1% BSA (w/v) in PBS), was applied and incubated
for 90 min at room temperature. After four washes with PBS, anti-mouse
IgG conjugated with FITC (Sigma) (1:50 dilution with 1% BSA (w/v) in
PBS) was then applied and reacted in the dark for 1 h at room
temperature. The cells were then washed 3 times with PBS, 2 times with
1% BSA (w/v), 1% Triton X-100 (v/v), PBS, and 3 times again with PBS.
The glass coverslips were mounted (Vectashield mounting medium for
fluorescence H-1000, Vector, Burlingame, CA), and photographs were
taken with incident illumination in a fluorescence microscope (Polyvar,
Reichert-Jung, Austria).
Conjugation of NLS Peptide with BSA
A peptide containing the SV40 large T antigen wild type NLS
CGGGPKKKRKVED with the N-terminal C providing a thiol group for conjugation and the GGG acting as a spacer was synthesized using Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry and
pentafluorophenyl ester on a PerSeptive 9050 synthesizer (PerSeptive
Biosystems, Hertford, UK) using standard protocols. Before conjugation,
BSA (5 µg/ml) was activated by incubation for 90 min at room
temperature in 100 mM HEPES-NaOH, pH 7.3, containing a
100-fold molar excess of sulfosuccinimidyl
4-[N-maleimidomethyl] cyclohexane 1-carboxylate (Pierce
and Warriner, Chester, UK) (19). Excess cross-linker was removed by gel
filtration on a PD10 column containing 9.1 ml of Sephadex-G25 (Amersham
Pharmacia Biotech). The activated BSA solution was added to a 30-fold
molar excess of NLS peptide. The pH was adjusted to 7.5, and the
reaction was allowed to proceed for 2 h at 37 °C. Uncoupled
peptide was removed by gel filtration on a PD10 column equilibrated in
150 mM NaCl. The molar ratio of coupling was 20-30
peptides/BSA molecule as estimated from the electrophoretic mobility.
For fluorescein conjugation,
carboxyfluorescein-N-hydroxysuccinimide ester (Boehringer
Mannheim) was dissolved in Me2SO and then added to
peptide-BSA solution in 0.2 M NaHCO3 at a 2:1
molar ratio to BSA. After overnight incubation at 4 °C, free
fluorescein was removed by gel filtration on a PD10 column. The
resulting conjugates were freeze-dried, dissolved in 150 mM
NaCl, and kept at Preparation of Cytosol Fractions for Nuclear Protein Import
Subconfluent HT29 cells (108) were collected by
scraping with a rubber policeman and washed twice with ice-cold PBS
followed by centrifugation for 5 min at 600 × g. The
cells were then washed once with 10 mM HEPES-HCl buffer (10 mM HEPES, pH 7.3, 10 mM potassium acetate, 2 mM magnesium acetate, and 2 mM dithiothreitol).
The cell pellet was gently resuspended in 1.5 volumes of 5 mM Hepes-HCl buffer (5 mM Hepes, pH 7.3, 10 mM potassium acetate, 2 mM magnesium acetate, 2 mM dithiothreitol, 20 mM cytochalasin B, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of
aprotinin and leupeptin) and kept for 15 min on ice before
homogenization by 30 strokes in a No. 2 bulb homogenizer (Jencons
Scientific Ltd. Leighton Buzzard, UK). The resulting homogenate was
centrifuged at 1,500 × g for 15 min to remove nuclei
and cell debris. The supernatant was then centrifuged at 100,000 × g for 30 min at 4 °C. The supernatant was dialyzed
extensively against transport buffer (20 mM Hepes-HCl, pH
7.3, 110 mM potassium acetate, 5 mM sodium
acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM dithiothreitol) and stored at Cell Permeabilization and in Vitro Nuclear Protein
Import--
Cell permeabilization and nuclear protein import was
carried out by a modification of the method described by Adam et
al. (20). HT29 cells cultured in glass chamber slides (Nunc,
Kamstrup, Denmark) were first rinsed in cold transport buffer and then
permeabilized in 40 µg/ml digitonin (Sigma) in transport buffer for 6 min at 4 °C. After extensive washes with ice-cold transport buffer,
transport mixture (final volume 140 ml) was applied (20% cytosol
fraction, 1 mM ATP, 5 mM creatine phosphate, 20 units/ml creatine phosphokinase, 2 mM BSA-NLS-fluorescein
in transport buffer). The chamber slides were maintained on ice during
the procedure. The protein transport reaction was started by
transferring the chamber slides into a 30 °C incubator and, after 30 min, was stopped by the addition of 1 ml of ice-cold transport buffer.
For the ABL inhibition experiment, before the addition of the
NLS-BSA-fluorescein, the permeabilized cells were first incubated with
ABL (0-100 µg/ml) for 15 min at room temperature in the presence of
cytosol fraction and ATP-regenerating system as above. Substitution of
wheat germ agglutinin (WGA, 100 µg/ml) for ABL acted as a positive control for this experiment. The cells were then washed extensively with transport buffer, and the slides were mounted and observed by
fluorescence microscopy (Polyvar, Reichert-Jung, Austria) with a
25× objective.
Effect of ABL on Mitogen-activated Protein Kinase (MAPK
(ERK-1 and ERK-2)) Nuclear Translocation
HT29 cells were cultured in 8-well glass chamber slides (Nunc)
for 24 h. After 1 wash with PBS, the cells were cultured in serum-free DMEM containing 250 µg/ml BSA for a further 24 h
before the addition of 40 µg/ml ABL (the same volume of PBS was added to control wells). Six h later, 20% FCS (v/v) was added to each well
for 20 min. The cells were quickly rinsed once with PBS and fixed in
10% paraformaldehyde (w/v) in PBS for 15 min at room temperature
followed by permeabilization with 100% methanol for 10 min at
Quantification of Relative Cytoplasmic and Nuclear
Fluorescence by Confocal Microscopy
Quantification of apparent nuclear and cytoplasmic fluorescent
ratios was achieved using Kinetic Imaging Lucida software (Kinetic Imaging, Liverpool, UK). From each cell in randomly chosen fields, a
2-µm-thick confocal slice centered on the nucleus was obtained. The
nucleus and whole cell sections were carefully traced using the
"lasso" function of the kinetic imaging software. At least 20 cells
were analyzed for each datum point.
Comparison of Effects of Free and Conjugated ABL on HT29 Cell
Proliferation
Twenty µg/ml free ABL produced 81% inhibition of thymidine
incorporation into DNA in HT29 colon cancer cells (Fig.
1). When ABL was immobilized by
conjugation to agarose, no significant inhibitory effect was seen at
concentration up to 100 µg/ml ABL (Fig. 1), suggesting that the
lectin has to be internalized to have its inhibitory effect on
proliferation. Minimum concentrations of ABL conjugates capable of
agglutinating type O human red blood cells were free ABL (2 µg/ml)
and FITC-ABL (4 µg/ml).
1-3GalNAc
(TF antigen)-binding
lectin (ABL) from the common edible mushroom (Agaricus
bisporus) has a potent anti-proliferative effect without any
apparent cytotoxicity. This unusual combination of properties prompted
investigation of its mechanism of action. In contrast to soluble
lectin, agarose-immobilized, and hence noninternalizable ABL had no
effect on proliferation of HT29 colon cancer cells. Electron microscopy
of HT29 cells incubated with fluorescein- and gold-conjugated ABL
showed internalization of the lectin into endocytotic vesicles and
multivesicular bodies. Confocal microscopy showed perinuclear
accumulation of fluorescein isothiocyanate-conjugated lectin, which
also inhibits HT29 cell proliferation, raising the possibility that the
lectin might interfere with nuclear pore function. Transport of heat
shock protein 70 into the nucleus in response to heat shock was blocked
by preincubation of HT29 cells for 6 h with 40 µg/ml ABL. In
digitonin-permeabilized cells, nuclear uptake of bovine albumin
conjugated to a nuclear localization sequence (NLS)-containing peptide
was also inhibited by a 15-min preincubation with 40-100 µg/ml ABL.
In contrast, serum-stimulated nuclear translocation of
mitogen-activated protein kinase, which is NLS-independent, was not
affected by pretreatment of cells with the lectin. These results
suggest that the anti-proliferative effect of ABL is likely to be a
consequence of the lectin trafficking to the nuclear periphery, where
it blocks NLS-dependent protein uptake into the nucleus.
INTRODUCTION
Top
Abstract
Introduction
References
1-3GalNAc
-),
is a common feature of malignant and premalignant epithelia
(5-7). In normal epithelia this structure is usually concealed (7),
probably by sialylation (8) or sulfation (9). We have previously shown
that noncytotoxic dietary lectins, which bind the TF antigen, are
capable of having marked proliferative and anti-proliferative effects
both in vitro and in vivo on normal and cancerous
human intestinal epithelial cells (10-14). Thus, stimulation of
proliferation has been shown with the TF-binding lectin, peanut
(Arachis hypogea) agglutinin (PNA) (10), as well as with
anti-TF monoclonal antibodies (15). In contrast, the lectin from the
common edible mushroom Agaricus bisporus (ABL), which unlike
PNA, can also bind to the 2-3 sialylated TF antigen, has been shown to
inhibit proliferation in a reversible and noncytotoxic fashion in a
wide range of epithelial cell types, blocking the growth-stimulatory
effects of epidermal growth factor, serum, and insulin (14). Because
many lectins are tightly globular proteins that are highly resistant to
heat and digestion (16) and can be detected in active form in feces
(10), their presence in foods is likely to be of considerable
importance in the functional relationship between diet and intestinal
proliferation and hence for intestinal cancer development (17). Because
ABL has a powerful anti-proliferative effect on malignant colon cells
without any apparent cytotoxicity, we have investigated the mechanism
by which this lectin elicits its biological effects.
EXPERIMENTAL PROCEDURES
80 °C.
80 °C. Protein
concentration was 10-20 mg/ml.
20 °C. The cells were blocked with 1% BSA (w/v) in PBS for 1 h and then incubated with 1.5 µg/ml rabbit polyclonal antibody to
phosphorylated MAPK (Promega Ltd, Southampton, UK) in 1% BSA (w/v),
0.25% Nonidet P-40 (v/v), PBS for 4 h (the same volume of 1% BSA
(w/v), 0.25% Nonidet P-40 (v/v), PBS was added to control cells but
without anti-MAPK antibody). After 6 washes with PBS, biotin-conjugated
anti-rabbit IgG (1:250 dilution in 1% BSA (w/v) in PBS) (Sigma) was
applied for 1 h followed by incubation of the cells with
avidin-FITC conjugate (1:200) (Sigma) for 1 h in the dark. After
washing with PBS, the cells were visualized and photographed using a
fluorescent microscope (Polyvar, Reichert-Jung, Austria) with a 25× objective.
RESULTS
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Fig. 1.
Comparison of the effects of free and
conjugated ABL on HT29 cell proliferation. A, cells were
cultured with 20 µg/ml ABL, 20 µg/ml (lectin concentration)
agarose-ABL, 20 µg/ml (lectin concentration) gold-ABL, 20 µg/ml
(lectin concentration) FITC-ABL, colloidal gold (same volume as
gold-ABL), FITC-BSA (20 µg/ml BSA) in serum-free DMEM containing 250 µg/ml BSA for 24 h before a 1-h pulse with 0.8 µCi/ml
[3H]thymidine. B, cells were cultured with
0-80 µg/ml ABL or 0-100 µg/ml (lectin concentration) agarose-ABL
in serum-free DMEM containing 250 µg/ml BSA for 24 h before a
1-h pulse with 0.8 µCi/ml [3H]thymidine. Values
represent mean ±S.D. of triplicate determinations from one of two
similar experiments expressed as a percentage of the incorporation of
[3H]thymidine observed in control cells (6530 ± 400 cpm).
As a prelude to electron and confocal microscopic studies of intracellular trafficking of the lectin, FITC- and gold-conjugated ABL were also assessed for their effects on agglutination and proliferation. Colloidal gold (10 nm)-conjugated-ABL (20 µg/ml ABL) had no significant effect on HT29 cell proliferation (Fig. 1), but at this concentration, the gold-ABL conjugate had only weak agglutinating ability (see below). Higher concentrations of gold-ABL were not attainable. In contrast, FITC-conjugated ABL (20 µg/ml ABL) produced 49% inhibition of HT29 cell proliferation (Fig. 1). The minimum concentration of these conjugates capable of agglutinating red blood cells was 16 µg/ml for both gold- and agarose-ABL. Neither 10-nm colloidal gold alone nor FITC-BSA (20 µg/ml BSA) had any effect on HT29 cell proliferation. We next examined the intracellular fate of gold-ABL and FITC-ABL.
Intracellular Trafficking of ABL
Electron Microscopy Studies-- Internalization of ABL by HT29 cells was demonstrated by electron microscopy in 10 separate experiments using 10-nm gold-conjugated ABL and also using fluorescein-conjugated ABL in view of the ability of the latter conjugate to inhibit proliferation. After incubation with HT29 cells for 1 h, gold-ABL was bound to the cell surface, and internalization had begun. The gold-ABL was aggregated in clathrin-coated pits and immediately beneath the plasma membrane in clathrin-coated vesicles (Fig. 2A). The cellular entry of gold-ABL also involved clathrin-independent pathways, because noncoated pits and pinocytotic vesicles also contained gold particles (Fig. 2A). A third mechanism of entry was macropinocytosis, recognized by association of the gold label with surface folds or villi and subsequent incorporation into vacuoles of 0.5 µm or more (result not shown). The gold-ABL extended beyond the system of early endosomes and was identified frequently within the endosomal carrier vesicles, often called multivesicular bodies (Fig. 2, B and D), which traffic to the late endosomal system (21). Over a 12-h incubation, more lectin became internalized, but it remained within membrane-bound vesicles that were often found in close proximity to the nuclear envelope. In control cells incubated with 10-nm colloidal gold, no gold was found inside the cells (results not shown). A further series of experiments with both 5- and 10-nm colloidal gold-conjugated ABL confirmed localization within the lumen of vesicles that were often close to the nucleus, but for both particle sizes, there was no clear evidence of lectin outside vesicles. Studies of the fluorescein-conjugated lectin (Fig. 2, C, D, and E) yielded almost identical results, with prominent accumulation within multivesicular bodies, although there was possibly some fluorescein-conjugated lectin identified within cytoplasm close to these bodies.
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Confocal Microscopy-- After a 1-h incubation of the cells with 20 µg/ml FITC-ABL, strong cell surface and intracellular fluorescence was observed (Fig. 3A). By 3 h, accumulation of fluorescence around the nucleus was seen in the majority of cells in each of 12 independent experiments (Fig. 3, B-D). In all cells, punctate intracellular FITC-ABL fluorescence was seen, but in addition, a ring of perinuclear fluorescence was observed. We then compared the intracellular trafficking of TRITC-dextran, a marker of fluid phase endocytosis (22), with that of FITC-ABL. Both conjugates were clearly internalized. However, the TRITC-dextran fluorescence was always punctate and showed no overlap with the distribution of FITC-ABL (Fig. 3E). These results suggested that FITC-ABL internalization is receptor-mediated rather than occurring via fluid phase endocytosis.
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Effect of ABL on the Nuclear Import of Heat Shock Protein 70 in Live Cells-- The perinuclear accumulation of FITC-ABL raised the possibility that the inhibitory effect of ABL on cell proliferation might be the consequence of interaction with the protein import machinery of the nuclear pores. To test this hypothesis, the effect of ABL on nuclear import of heat shock protein 70 (Hsp70) was studied. Hsp70 is produced by prokaryotic and eukaryotic cells in response to environmental changes such as heat stress. There are two forms of Hsp70, one (Hsp73) is constitutively expressed, whereas the synthesis of the other (Hsp72) is induced by heat shock from existing mRNAs (23). During heat stress, both forms of the Hsp70 are imported into the nucleus, whereas a small quantity remains cytoplasmic (18, 23). During recovery from heat stress, Hsp70 leaves the nucleus and becomes distributed throughout the cytoplasm (18).
As expected, Hsp73 became concentrated in the nuclei of the control cells after 1 h of heat shock at 43 °C (Fig. 4A , a). However, when the cells were pretreated with 40 µg/ml ABL for 6 h, Hsp73 remained in the cytoplasm after the same heat shock (Fig. 4A, b). To rule out the possibility that this effect might be because of the inhibition of Hsp73 synthesis by ABL, a parallel experiment was performed in which the cells were cultured in the presence or absence of ABL and lysed after 1 h of heat shock at 43 °C, and immunoblots were performed with anti-Hsp73. This demonstrated that ABL had no effect on the cellular content of Hsp73 (Fig. 4B). Pretreatment of the cells with the growth stimulatory TF-binding peanut lectin (PNA), did not have any effect on Hsp73 nuclear translocation (Fig. 4A, c).
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Further experiments were performed with an anti-Hsp70 monoclonal antibody specific for the heat-inducible form of Hsp70 (Hsp72). Using this antibody, Hsp72 in non-heat-shocked cells was found only in the cell cytoplasm (Fig. 5A, a). After heat treatment at 43 °C for 3 h, Hsp72 accumulated inside the nucleus (Fig. 5A, b). Pretreatment of the HT29 cells with 40 µg/ml ABL for 6 h again excluded the entry of Hsp72 into the nucleus (Fig. 5A, c), whereas 40 µg/ml PNA pretreatment of the cells for 6 h again did not affect Hsp72 subcellular distribution (Fig. 5A, d). A similar increase of Hsp72 expression after heat treatment occurred in both the ABL-treated and untreated cells, suggesting that ABL has no effect on the induction of Hsp72 by heat shock (Fig. 5B). Quantification of the cellular distribution of Hsp72 using confocal microscopy shows that the inhibition of Hsp72 nuclear import by ABL is both dose- and time-dependent (Fig. 5, C and E). The maximal effect produced by 80 µg/ml ABL occurred after a 6-h incubation with the cells. This effect of ABL on Hsp72 nuclear import was abolished by the presence of 100 µg/ml TF-expressing asialo bovine mucin (Fig. 5D).
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Effect of ABL on Nuclear Import of NLS-BSA Fluorescein in Digitonin-permeabilized Cells
The observation that ABL inhibits the nuclear import of Hsp70
suggested that this lectin may have a more general effect on protein
transport into the nucleus. For many large proteins, import into the
nucleus requires the presence in the protein of a NLS (24). An
essential first step in nuclear import of such proteins is the binding
of this sequence by NLS receptors on the cytoplasmic protein, importin
(25). To assess the effect of ABL on NLS-dependent nuclear protein import, a synthetic peptide containing the SV40 large T
antigen wild type NLS (CGGGPKKKRKVED) (24, 26) was
directionally conjugated through the thiol group of the N-terminal C to
BSA. The conjugate was labeled with fluorescein and employed to assess
nuclear import in digitonin-permeabilized cells, as described by Adam
et al. (20). In the absence of lectin, NLS-BSA fluorescein
was transported into the cell nucleus after 30 min (Fig.
6a). In three separate
experiments, when permeabilized cells were pretreated for 15 min with
100 µg/ml ABL, a marked inhibition of nuclear import of the NLS-BSA
fluorescein was observed (Fig. 6c). Substitution of WGA (100 µg/ml) for ABL acted as a positive control for this experiment (Fig.
6b). No direct binding of ABL to NLS-BSA fluorescein could
be demonstrated in a control experiment in which the NLS-BSA (30 µg)
was subjected to a 10% SDS-polyacrylamide gel electrophoresis,
transferred to nitrocellulose membrane, and blotted with
peroxidase-conjugated ABL (10 µg/ml) (results not shown).
Quantification analysis of NLS-BSA nuclear import by confocal microscopy again demonstrated a dose-dependent effect of
ABL, and the effect of 100 µg/ml ABL on NLS-BSA nuclear protein
import could be completely abolished by the presence of 100 µg/ml
asialo bovine mucin (Fig. 6d).
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Effect of ABL on MAPK Nuclear Translocation-- It has recently been recognized that some large proteins enter the nucleus by NLS-independent mechanisms (27). For example, the nuclear import of MAPK, which does not possess a NLS, is not affected by WGA, confirming a non NLS-dependent nuclear import process (28). In control cells, after a 20-min stimulation with 20% FCS, immunoreactive MAPK was localized mainly in the nucleus (Fig. 7a). The nuclear accumulation of MAPK was not affected by a 6-h pretreatment of the cells with 30 µg/ml ABL (Fig. 7b).
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Effects of ABL on DNA, RNA, and Protein Synthesis-- The effect of ABL on DNA, RNA, and protein synthesis in HT29 cells was assessed by measuring the incorporation of [3H]thymidine, [3H]uridine, and [3H]leucine into trichloroacetic acid -precipitable macromolecules. Although the synthesis of DNA was almost completely inhibited after 24 h of incubation with 20 µg/ml ABL, the inhibition of RNA synthesis and protein synthesis was much less marked (Fig. 8). This continued ability of HT29 cells to synthesize RNA and protein reflects the noncytotoxic inhibition of cell growth by ABL and confirms that NLS-specific blockade of nuclear protein import by ABL does not result in cell death.
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DISCUSSION |
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A. bisporus lectin ABL inhibits cell proliferation in a wide range of cells without cytotoxicity (14), suggesting that this lectin might affect a cellular process fundamental to cell division. In the present study, we found that agarose-conjugated ABL is not anti-proliferative, indicating that ABL has to be internalized to inhibit HT29 cell proliferation. Internalization of the lectin was directly demonstrated with both gold- and FITC-conjugated ABL. Both gold-ABL and FITC-ABL were predominantly associated with the lumen of vesicles and were both present within multivesicular bodies, many of which were in close proximity to the nuclear membrane (Figs. 2 and 3). Some FITC-ABL but not gold-ABL was apparently in cytoplasm around the multivesicular bodies, but it is not clear to what extent this difference between the lectin conjugates might have been the consequence of the different fixation processes used for the electron microscopy sections. The FITC-ABL but not the gold-ABL was shown to inhibit cell proliferation.
The demonstration by confocal microscopy that internalized FITC-ABL accumulated around the nucleus (Fig. 3) led us to speculate that the lectin might interfere with nuclear pores, perhaps blocking nuclear protein import. In live HT29 cells, preincubation with ABL inhibited nuclear import of Hsp70 in response to heat shock (Figs. 4 and 5). Hsp70 has itself been shown to be an essential component of the mechanism for NLS-dependent nuclear protein import (29-32), possibly as a molecular chaperone to promote the formation and stability of the NLS cargo complex (32). Inhibition by ABL of nuclear import of synthetic NLS peptide in permeabilized HT29 cells (Fig. 6) provides further evidence that ABL inhibits the NLS-dependent nuclear protein import mechanism. Inhibition of nuclear protein transport has previously been demonstrated with WGA and Sambucus nigra lectins but only in digitonin-permeabilized cells or following direct injection of the lectin into cells or isolated nuclei (33-35).
Molecular exchanges between the nucleus and cytoplasm occurring via the
nuclear pore complexes (36, 37) are fundamentally important for cell
growth. The nuclear pore complexes accommodate both passive diffusion
of ions and globular proteins of less than 20 kDa (38, 39) and active
transport of larger macromolecules (36, 37) through a gated channel by
sequence-dependent (40, 41), and
energy-dependent mechanisms (42, 43). Protein transport into the nucleus is multistepped and includes the binding of the transport ligand by NLS receptors and movement of the transport ligand-NLS receptors to the nuclear pores (25). Cytosolic factors such
as the NLS receptor/importins (44, 45), p97 (46), the GTPase Ran/TC4
(47, 48), Hsp70 (29, 30), and NTF2/B-2 (49) have been shown to be
essential for this process. After GTP hydrolysis by Ran at the nuclear
pore complex binding site, the receptor-ligand-Ran.GDP complex is
translocated into the nucleus. This involves interactions of the
complex with the O-GlcNAc glycosylated glycoprotein p62
localized on both the nucleoplasmic and cytoplasmic side of the nuclear
pore complexes (50-52). Binding to p62 by monoclonal antibody (53),
WGA (GlcNAc binding) (33-35), and S. nigra agglutinin (sialic acid 2-6 Gal/GalNAc binding) (35) all inhibit protein transport into the nucleus. Several pieces of evidence suggest, however, that ABL is unlikely to be acting via direct interaction with
p62. ABL has been shown not to bind N-acetyl-glucosamine (54), and the absence of staining of the nuclear periphery by FITC-conjugated peanut lectin (33) implies the absence of unsialylated Gal1-3GalNAc
- at this site. Moreover, although sialic acid has recently been demonstrated on p62, its enzymatic removal does not
reveal the peanut lectin receptor (55), suggesting that sialyl 2-3
Gal
1-3GalNAc
, the alternative ligand for ABL, is also absent
from p62.
Alternatively, ABL may bind to a cytosolic component, which is
essential for nuclear protein import, e.g. p97, importin or
, RanBP2, or NTF2. This also raises the possibility that the ligand for ABL may also be a ligand for one of the naturally occurring intracellular galactose-binding lectins (galectins) (56). However, no
intracellular
-galactoside-containing glycoproteins have been identified so far, and it is also possible that ABL may interact with
its ligand by protein-protein interaction, as it has been shown that
peptides may mimic galactosides and interact with the carbohydrate
recognition domain of galactose-binding lectins (57). We do not yet
have clear evidence that the lectin gains access to the cytosol, and an
alternative possibility is that it is exerting its effect by
interaction with the luminal side of a transmembrane glycoprotein.
It is now apparent that there are at least three different mechanisms for nuclear import of large proteins; first, the NLS-dependent mechanism, which has been well characterized (43, 52); second, a WGA-inhibitable but non-NLS-dependent mechanism described for nuclear import of heterogeneous nuclear ribonucleoprotein via a 38-amino acid M9 domain (58) interacting with transportin (27); and third, the non-WGA-inhibitable non-NLS-dependent mechanism described for nuclear import of MAPK (28). It seems likely that the inhibition of NLS-dependent nuclear import by ABL is relevant to its effect on proliferation, whereas its lack of effect on the MAPK import pathway (Fig. 7) may explain why the cells can still survive after inhibition of the classical NLS-dependent pathway. Previous studies have shown that intracytoplasmic injection of anti-Hsp73 antibody also blocks NLS-dependent nuclear protein import without causing cell death (29).
The present study suggests that inhibition of cell proliferation by ABL
is a consequence of the specific trafficking of the lectin to the
nuclear periphery where it blocks NLS-dependent protein
uptake into the nucleus. Further studies are in progress to
characterize the intracellular ABL-binding glycoproteins and identify
the molecular target of ABL in the NLS-dependent nuclear transport machinery.
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FOOTNOTES |
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* This work was supported by grants from the North West of United Kingdom Cancer Research Fund (to L.-G. Y., D. G. F., D. G. S., R. C. E., and J. M. R.), a program grant from the Medical Research Council (to O. V. G. and O. H. P.), and equipment grants from Biotechnology and Biological Sciences Research Council, Higher Education Funding Council for England and Wales, Arthritis Research Campaign and the North West Regional Health Authority (to M. R. H. W.). Confocal microscopy work was supported through collaboration with Carl Zeiss Ltd.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. Tel.: + 44 (0)151-7064073; Fax: + 44 (0)151-7065802; E-mail:
rhodesjm{at}liv.ac.uk.
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ABBREVIATIONS |
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The abbreviations used are:
TF antigen, Thomsen-Friedenreich antigen (Gal1-3GalNAc
-);
ABL, A.
bisporus lectin;
BSA, bovine serum albumin;
FCS, fetal calf serum;
FITC, fluorescein isothiocyanate;
Hsp70, heat shock protein 70;
MAPK
(ERK-1 and ERK-2), mitogen-activated protein kinase;
NLS, nuclear
localization sequence;
NTF2, nuclear transport factor 2;
PNA, A.
hypogea lectin;
WGA, wheat germ agglutinin;
DMEM, Dulbecco's
modified Eagle's medium;
PBS, phosphate-buffered saline;
TRITC, tetramethylrhodamine isothiocyanate.
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REFERENCES |
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