1 Max-Planck-Institute of Biophysical Chemistry, Department of Neurobiology,
Göttingen, Germany
2 Engelhardt Institute of Molecular Biology, Department of Molecular Biology,
Vavilova Street 32, Moscow, Russia and The Oslo University Center for Medical
Studies at Moscow, Vasilova Street 34/5, Moscow, Russia
3 Medical Institute, Minsk, Belorussia
* Authors for correspondence (e-mail: imajoul{at}gwdg.de ; hsoelin{at}gwdg.de )
Accepted 5 November 2001
![]() |
Summary |
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Key words: AB5-toxins, Glycosphingolipid receptors, Cell cycle, Golgi
![]() |
Introduction |
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Cell differentiation affects synthesis and plasma membrane expression of
Gb3. Undifferentiated THP-1 cells express significantly more Gb3 on their
surface than differentiated cells
(Ramegowda et al., 1996),
while the situation is opposite in intestinal epithelial cells where
differentiated cells express high levels of Gb3 on their surface and
undifferentiated cells show an almost complete loss of Gb3 (Jacewicz et al.,
1975). In some cells the expression of Gb3 on the cell surface seems to be
developmentally regulated. In rabbit intestinal microvilli epithelial cells,
the synthesis of Gb3 increases during the third week of life together with an
increased sensitivity to the toxic effects of ST (Mobassaleh et al., 1994).
The cellular level of gangliosides also seems to be regulated by
differentiation. It decreases in 3T3-L1-adipocytes as they become
differentiated. In particular, the level of GM1 drops by 75-80%. These changes
could not be observed in the non-differentiating 3T3-C2 cell line
(Reed et al., 1980
). In colon
carcinoma cells (Nojiri et al.,
1999
) as well as in renal carcinoma cells
(Saito et al., 2000
), the
synthesis of ganglioside GM3, a precursor of GM1, increases following
induction of differentiation. The percentage of cultivated MFII-fibroblasts
exhibiting high levels of GM3 increases as cells become confluent
(Rosner et al., 1990
). A
similar observation has been made with cultivated Zejdela hepatoma cells
(Staedel-Flaig et al., 1987
).
Interestingly, treatment of a variety of different cell types with butyrate
induces synthesis and cell surface expression of GM1 as well as of Gb3
(Fishman and Attikan, 1979), leading to an enhanced binding of CTX as well as
of ST and verotoxin. Choi et al. (Choi et
al., 1997
) have studied the levels of neutral and acidic
gangliosides in different phases of the cell cycle in a glioma cell line.
Cell-cycle-dependent changes, in particular of GM3, GM2 and GM1, could not be
observed, although a decrease in b-series gangliosides was measured during
metaphase.
We report here that Vero cells, a green monkey kidney tumor cell line, show cell-cycle-dependent but inverse changes in surface-expressed GM1 and Gb3. During interphase and G1, cells bind almost exclusively CTX; during S-phase binding of both CTX and ST is very low. During G2, binding of ST increases and remains high during metaphase and telophase, whereas the binding of CTX remains low until G0/G1 is reached again. The cell-cycle-dependent changes in the expression of GM1 and Gb3 on the cell surface result from corresponding changes of synthesis of GM1 and Gb3, respectively. These observations were confirmed also for other cell types including Vero 317 cells, undifferentiated and differentiated PC12 cells and astrocytes, suggesting that a conserved mechanism is underlying this novel phenomenon.
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Materials and Methods |
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Toxins and antibodies
CTX-K63 (kindly donated by Mariagrazia Pizza, Siena, Italy) was used
throughout the experiments. CTX-K63 has a S63K-mutation in the
CTX-A subunit that completely abolishes its ADP-ribosylating activity
(Fontana et al., 2000) without
affecting its intracellular transport
(Majoul et al., 1998
). ST was
isolated as described previously (Kozlov
et al., 1993
) (from the Engelhardt Institute of Molecular Biology,
Moscow). Both toxins were directly labeled with Cy3- or Cy5-monosuccinimidyl
esters or with Cy2-bissucccinimidyl ester according to the Amersham Pharmacia
Biotech protocol. Labeled proteins were separated from free chromophores by
gel filtration over G-10 and concentrated by Centricon-10 (AmiconTM)
centrifugation. The labeling stoichiometry was determined from the absorptions
at 280 nm (protein), 487 nm (Cy2), 550 nm (Cy3), or 650 nm (Cy5). The molar
dye:protein ratios ranged from 0.2 to 1. A polyclonal rabbit anti-cyclin B1
antibody and a mouse monoclonal anti-BrdU-antibody came from Transduction
Laboratories (BD Biosciences, Heidelberg, Germany), and a FITC-conjugated
mouse monoclonal anti-BrdU-antibody was from Pharmingen (BD Biosciences,
Heidelberg, Germany). Mouse monoclonal anti-cyclin B1 antibodies were a kind
gift of Jonathan Pines (CRC, Cambridge, UK). A
horseradish-peroxidase-conjugated goat anti-rabbit antibody was from Zymed
Laboratories (San Francisco, USA).
Cell cultures
Vero, Vero 317 and PC 12 cells, mouse fetal astrocytes and hippocampal
neurons of newborn mice were cultivated in DMEM plus 10% FCS supplemented with
100 U/ml penicillin G and 0.1 mg/ml streptomycin. PC12 cells were cultivated
on collagen (Vitrogen) or poly-L-lysine (Sigma) coated cover slips;
hippocampal neurons were cultured on a feeder layer of fetal mouse astrocytes.
For the toxin binding experiments, cells were transferred to DMEM without
phenol red, buffered with 10 mM Hepes.
Determination of cell cycle phases
S-phase cells were identified by the incorporation of BrdU. To this end,
Vero cells were incubated for 30 minutes under standard conditions in the
presence of 50-100 µM BrdU. Cells were washed with PBS, cooled on ice and
exposed to Cy-dye-labeled ST and CTX as below. Cells were fixed with 4%
paraformaldehyde and permeabilized with 0.1% saponin. Following blocking with
PBS/0.1% fish gelatine, the incorporation of BrdU into DNA was detected by
immunofluorescence using either a BrdU-specific monoclonal antibody (1/100) in
combination with Cy3- or Cy5-labeled rabbit-anti-mouse secondary antibodies
(1/200) or a FITC-conjugated monoclonal anti-BrdU antibody (1/100).
To identify cells in G2 or metaphase, the expression and intracellular distribution of endogenous cyclin B1 was analysed by immunofluorescence using a polyclonal rabbit anti-cyclin B1-antibody or a mouse monoclonal anti-cyclin B1 antibody. For detection Cy-dye labeled secondary antibodies were used. DNA was detected with DAPI dissolved in Moviol.
Synchronization and cell cycle arrest
To obtain G0/G1 phase Vero cells, cells were maintained in 0.1% FCS for 36
hours. After addition of serum within 8-10 hours, more than 30% of Vero cells
were in G2 phase. For differentiation of PC12 cells and neurons, 100 ng/ml NGF
was added to the culture medium.
For synchronization by a double-thymidine block the exponentially growing Vero cells were arrested in G1 by treatment with thymidine (3 mM) at 0.1% FCS. After 12 hours, cells were released from the block by washing with PBS and addition of fresh medium (DMEM, 10% FCS). Cells were grown for another 10 hours and treated a second time with 3 mM thymidine for 12 hours followed by release from the block (as with the first block). Twelve hours after this release, 30-50% of cells revealed a mitotic phenotype and nuclear staining for cyclin B1. Binding of the toxins to the cell monolayer was analyzed 3, 6, 12, and 24 hours after the release from the second thymidine block.
Alternatively, cell monolayers were treated after 12 hours in culture with 5 µg/ml colchicine for 2 or 3 hours. Detached cells were sedimented by centrifugation (800 g for 5 minutes), washed with PBS and exposed to Cy-dye-labeled toxins at 0°C while in suspension. The cells were washed again and sedimented by centrifugation. An aliquot of the sedimented cells was plated on a cover slip for fluorescence microscopy.
Binding of Cy-labeled Shiga and Cholera toxin to cell surface
receptors
The binding conditions for Shiga and Cholera toxin were similar to those
described previously (Majoul et al.,
1996). In short, cells were exposed to 0.5 µg/ml each of CTX
and/or ST at 0°C for 15 minutes. Unbound toxins were removed by washing at
0°C. The temperature was then increased to 25°C for 5 minutes.
Immediately thereafter the live cells were analyzed by fluorescence
microscopy. In this way, toxin binding as well as initial toxin uptake could
be analyzed.
Immunoblotting of cyclin B1 from FACS-separated Vero cells
Non-synchronized Vero cells were exposed to Cy-dye labeled ST and CTX as
described above. Cells separated by FACS as described below were lysed on ice
in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1%
Triton X-100 with 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 50 µM
PMSF). Aliquots from the lysate were heated for 5 minutes in Laemmli sample
buffer, resolved by 12% SDS-PAGE and electroblotted onto a PVDF membrane
(ImmobilonTM). The membranes were blocked in 2% dry milk in PBS and
incubated with a polyclonal rabbit anti-cyclin B1 antibody (1/1000) followed
by chemoluminescence detection with a horseradish-peroxidase-coupled secondary
antibody (ECL system, Amersham-Pharmacia, Freiburg). Antibodies were removed
by incubation in 0.1 M citric acid, pH 3.3. The blot was then probed a second
time with a rabbit antiprotein disulfide isomerase antibody (1/500) and
detected by chemoluminescence using the same secondary
horseradish-peroxidase-labeled goat anti-rabbit antibody as above.
Analyses of intracellular receptors for Cholera and Shiga toxin
Non-synchronized Vero cells were incubated at 0°C with either Cy3-ST or
Cy5-CTX (0.5 µg/ml) for 20 minutes. Cells were washed at 0°C to remove
unbound toxins followed by fixation with 4% paraformaldehyde for 10 minutes.
Thereafter cells were exposed to the same concentration of unlabeled toxins at
0°C for 5 minutes to block the remaining toxin receptors on the cell
surface; they were then washed and fixed again with 4% paraformaldehyde for 5
minutes. Remaining aldehyde groups were blocked with 50 mM NH4Cl.
Cells were then permeabilized with 0.1% (w/v) saponin in PBS/0.1% fish gelatin
and stained with Cy-3 or Cy5-labeled toxins for 20 minutes at room
temperature. Unbound toxins were removed by washing with PBS, and cells were
analyzed by fluorescence microscopy.
Fluorescence and immunofluorescence microscopy and image
processing
When binding and uptake of Cy-dye labeled toxins was analyzed in live
cells, the cells remained in phenol red free DMEM with 5-7% FCS. Fluorescence
microscopy was performed with a Zeiss Axioplan microscope using 63x Plan
Achromat and 100x Neofluar objectives and filters for excitation at
ex=488 nm for Cy2 fluoresence and
ex=514
nm for Cy3 fluorescence.
Fixed cells, as used during the analysis of cell cycle markers (see above), were observed using a filter set for AMCA, FITC and Cy3. Simultaneous detection of Cy2- and Cy3-labeled samples was performed with a Zeiss LSM 410 laser scan microscope using laser excitation at 488 nm and 514 nm respectively. Cy5 was detected and laser excitation at 633 nm.
Differential display of Cholera and Shiga toxin bound cells
Non-synchronized Vero cells were plated on 1.4 cm culture dishes as
described above. After 12 hours, cells were washed with PBS and the dishes
transferred to ice. Cells were treated for 20 minutes at 0°C with Cy2-ST
and Cy3-CTX (0.2 µg/ml each). Unbound toxins were removed by washing with
ice-cold PBS. Cells were then detached by incubation with PBS containing 10 mM
EDTA. Cy2- and Cy3-labeled cells were separated using a BD FACSVantage SE flow
cytometry system (BD Biosciences), using filter settings for FITC and TRITC.
RNA was extracted from the separated cells according to Chomczynski and Sacchi
(Chomczynski and Sacchi, 1987)
using the peqGOLD RNA PureTM reagent and the protocol of the supplier
(Peqlab, Erlangen, Germany). cDNA was synthesized using oligodT primers and
MMLV reverse transcriptase (DeltaTM RNA Fingerprinting Kit, Clontech,
Heidelberg, Germany). For amplification, ten 5'-primers (P1-P10) and
nine 3'-primers (T1-T9) were used (DeltaTM RNA Fingerprintin Kit,
Clontech, Heidelberg, Germany) together with Advantage Klen Taq
polymerase in the presence of [
-32P]-ATP. The first cycle
was run for 5 minutes at 94°C, 40°C and 72°C; the following two
cycles were run for 2 minutes at 94°C and for 5 minutes each at 40°C
and 72°C. This was followed by 23 cycles run for 1 minute each at 94°C
and 60°C and 2 minutes at 72°C. The PCR products were separated by
electrophoresis on denaturing 5% polycrylamide gels. The gels were blotted
onto Whatman 1 paper, vacuum-dried and autoradiographed using Kodak MR film.
Differentially amplified cDNA-fragments were cut out from the gel and
reamplified under high stringency conditions with the P/T-primer combination
used for differential display. The amplified fragments were cloned using the
TOPOTM-TA cloning kit (Invitrogen), amplified and sequenced. The
possibility of contamination by somatic DNA was excluded by comparing the band
patterns of non-transcribed RNA-samples with those of RNA-free control
samples; both gave identical results. Moreover, using the method described,
intron sequences were never detected.
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Results |
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Transcription is differently regulated in Shiga-toxin- and
Cholera-toxin-binding cells
Vero cells were incubated with Cy2-ST and Cy3-CTX and separated by FACS.
RNA from the separated cells was extracted, reversed transcribed and analyzed
by differential display. The pattern of DNA fragments showed distinct
differences between ST- and CTX-binding cells
(Fig. 1b). Although the cDNAs
corresponding to the RNA fragments have not been identified yet, the different
pattern shows clearly that the differences between the two cell populations is
not restricted to their differential toxin binding.
The different toxin binding patterns correspond to different phases
of the cell cycle
When Vero cells were diluted to the single cell level and recultivated to
obtain clonal cells, again a mixture of ST- and CTX-binding cells was obtained
after 24-48 hours (data not shown), which indicates that the different toxin
binding patterns did not represent genetically different cells but rather
different states of clonal cells. This led us to test the hypothesis that the
differences in the binding of the two toxins might reflect different phases of
the cell cycle. The following experiments were carried out to test this
hypothesis.
Cultured cells in metaphase bind weakly to cover slips and can, therefore, be detached by colchicine treatment and shaking, whereas cells in interphase or G1 remain on the glass under the same condition. Therefore, Vero cells were treated with colchicine as described in the Methods section, and detached cells and cells that remained bound to the cover slip were analyzed for ST- and CTX-binding. The detached and replated cells had a round-shaped appearance and bound exclusively to ST (Fig. 2a), whereas most of the cells remaining on the cover slip were larger, flat and irregularly shaped and bound only to CTX (Fig. 2a). Of 150 detached and replated cells, all showed a strong binding of ST, whereas only three of these cells exhibited a significant binding of CTX. On the other hand, of the 50 large, flat, irregularly shaped cells that had remained on the coverslip after colchicine treatment and shaking, all bound strongly to CTX, and none of these cells exhibited a significant binding of ST. When the detached cells, which bound almost exclusively to ST, were reseeded, cultured in the presence of serum for 12 hours and then analyzed for toxin binding, the normal distribution between CTX- and ST-binding cells was obtained (Fig. 2b). These results indicate that Vero cells in interphase or G1 bind preferentially to CTX, whereas cells in metaphase or anaphase bind mainly to ST.
|
To further substantiate this conclusion, Vero cells were partially synchronized by a double thymidine block. Twelve hours after release from the block, 30-50% of the cells were in metaphase or anaphase (Fig. 3). These cells bound almost exclusively to ST (Fig. 3, upper left panel), whereas cells in interphase or G1, which were characterized by their strong adhesion and their flat and extended appearence, bound only to CTX (Fig. 3, upper right panel).
|
In order to obtain a more detailed picture, we correlated the binding of
the two toxins with the incorporation of BrdU on one hand and the expression
and intracellular distribution of cyclin B1 on the other. Strong incorporation
of BrdU is an indicator of S-phase, whereas an increased expression and an
enhanced nuclear uptake of cyclin B1 is an indicator for G2 and metaphase
(Kakino et al., 1996;
Jin et al., 1998
). As depicted
in Fig. 4, cells in S-phase
exhibited only low binding of both toxins, indicating that the expression of
receptors for both toxins on the cell surface strongly decreases during
S-phase. All of the 50 cells showing a strong incorporation of BrDU exhibited
either no or a very low binding to CTX and ST. When the degree of toxin
binding was qualified using a 7 degree scale (ranging from 1 equals no
detectable binding to 7 equals very strong binding) the mean binding of CTX
was 2.3±1.1 and the mean binding of ST was 2.2±1.3. As the
binding of CTX remained low through S-phase, G2, metaphase and anaphase (see
below) it is clear that some BrDU-negative cells show a low degree of
CTX-binding.
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When cells move from S-phase into G2 and metaphase, the expression of cyclin B1 increases together with an enhanced translocation into the nucleus (Fig. 5a,b). This shift was associated with an increase in the binding of ST, whereas the binding of CTX remained as low as during S-phase (Fig. 5a,b). Out of the 60 cells that gave a strong nuclear signal for cyclin B1, all showed a strong binding of ST, whereas binding of CTX to these cells was either undetectable or very low. Those cells in which significant amounts of cyclin B1 were detected in the nucleus, showed a very strong binding of ST. This was further substantiated by estimating the amount of cyclin B1 from immunoblots from ST- and CTX-positive cells after their separation by FACS (Fig. 5c). Clearly, the cells preferentially binding to ST expressed significantly more cyclin B1 than the CTX-positive cells.
|
In summary, we find that cells in interphase and G1 bind almost exclusively to CT. Cells in S-phase exhibit a decreased binding of both toxins, whereas the transition from S-phase to the G2, metaphase and anaphase is associated with a loss of binding of CTX and an increased binding of ST (Fig. 5d). Upon transition into G1, binding of CTX reappears, whereas the ability of the cells to bind to ST decreases or disappears.
Decreased toxin binding results from decreased receptor
biosynthesis
As binding of the two toxins to the plasma membrane is determined by the
density of receptor molecules in the membrane, we conclude that the
cell-cycle-dependent differential binding of the two toxins reflects
cell-cycle-dependent expression of GM1 and Gb3 on the plasma membrane. This
could result from reduced or absent synthesis of toxin receptors or from a
block of receptor transport from the place of synthesis to the plasma
membrane. Therefore, we analyzed the expression of intracellular receptors in
permeabilized Vero cells. Unpermeabilized cells showing strong binding to CTX
also exhibited significant binding of this toxin to intracellular sites
(Fig. 6a). The same holds for
cells showing strong surface binding of ST. Cells that exhibited no or little
binding of either toxin to the cell surface were also devoid of intracellular
toxin binding sites for the corresponding toxin after cell permeabilization
(Fig. 6a,b). This allows for
the conclusion that changes in the binding of either toxin results from the
corresponding changes in synthesis of the glycolipid receptors.
|
Cell-cycle-dependent binding of Cholera toxin to Vero 317 cells
Both the ganglioside GM1 and the globoside Gb3 are
synthesized from the same precursor, namely lactosyl ceramide. We, therefore,
explored the possibility that an increased synthesis of Gb3 from
lactosylceramide during late G2 and M-phase might have competed for the
synthesis of GM3 or GA2, precursors of GM1
(Fig. 10). This hypothesis was
tested in Vero 317 cells, which are unable to bind ST
(Pudymaitis et al., 1991)
while still binding CTX (Fig.
7). As these cells also show a decreased or absent binding of ST
after permeabilization of the cells in comparison to normal Vero cells (data
not shown), the decreased surface binding of ST by Vero 317 cells indicates
not simply a disturbed translocation of Gb3 to the plasma membrane but a
decreased synthesis of Gb3. Therefore, if the decreased binding of CTX during
S-phase, G2 and M-phase in normal Vero cells results from a competition for
lactosylceramide owing to increased Gb3 synthesis, such a decrease should not
occur in Vero 317 cells. We analyzed, therefore, the cell-cycle-dependent
binding of CTX in Vero 317 cells.
|
|
Similar to normal Vero cells, Vero 317 cells show CTX binding in flat, extended cells of the G0/G1-type (Fig. 7). Vero 317 cells in S-phase, which are characterized by a high incorporation of BrdU, bound little or no CTX (Fig. 7). From this we conclude that the cell-cycle-dependent changes in expression of the CTX-receptor GM1 on the cell surface does not result from a cell-cycle-dependent competition for lactosylceramide. This conclusion is supported by data obtained from PC12 cells and from astrocytes (see below).
Cell-cycle-dependent binding of CTX to PC12 cells, primary cultured
hippocampal neurons and astrocytes
We next addressed the question of whether the cell-cycle-dependent
expression of toxin receptors is restricted to Vero and Vero 317 cells or
whether it occurs also in completely different cell types.
To this end, we performed experiments with PC12 cells, a neuro-endocrine
cell line. PC12 cells bind CTX but not ST
(Fig. 8a,c). But not all PC12
cells showed strong CTX binding; many cells exhibited weak or no CTX binding.
In particular PC12 cells in S-phase, as indicated by a high incorporation of
BrdU, did not bind CTX (Fig.
8h,i). In order to achieve some kind of synchronization, we
treated PC12 cells with NGF, as this growth factor is known to induce cellular
differentiation and arrest of the cells in G0/G1
(Hughes et al., 2000). Indeed,
all cells showing signs of differentiation (flat, extended shape, development
of axonal protrusion or axons) bound CTX
(Fig. 8d,g). This indicates
that in PC12 cells, CTX binding is regulated by the cell cycle in a similar
way as in Vero and Vero 317 cells. This conclusion was further supported by
our observation that primary cultured hippocampal neuronal cells, which exist
only in interphase, uniformly exhibited a strong binding to CTX
(Fig. 9d,e).
|
|
As the hippocampal neuronal cells were cultivated on top of a feeder layer of astrocytes, we have also explored the binding of the two toxins to these cells. Astrocytes bound ST as well as CTX, but, similar to Vero cells, cells exhibiting a strong binding of ST did not bind CTX and vice versa (Fig. 9a,c). Furthermore, astrocytes in S-phase, as indicated by a strong incorporation of BrdU, showed little binding of both toxins (Fig. 9b,c). Thus, astrocytes show the same cell-cycle-dependent differential expression of receptors for ST and CTX as Vero cells.
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Discussion |
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A decreased expression of these glycolipids on the cell surface could
reflect either their decreased net synthesis or an inhibition of their
translocation from the Golgi to the plasma membrane. An enhanced degradation
of the glycolipids or their increased endocytosis could also be responsible.
We observed that cells that did not bind ST or CTX on the outer plasma
membrane had also lost the corresponding binding sites inside the cell. This
argues strongly for an increased net synthesis of Gb3 or GM1 in cells
exhibiting an increased binding of ST or CTX, respectively. Using verotoxin, a
Shiga-like toxin, in experiments with Vero cells, Pudymaitis and Lingwood
(Pudymaitis and Lingwood,
1992) observed a maximum toxicity at the G1/S-phase boundary but
could not detect changes in the cellular Gb3 content or in the incorporation
of [14C]-galactose into Gb3. In these experiments, the whole
population of cultured cells was analyzed at different time points after a
double-thymidine block. Thus, a correlation between surface expression of Gb3
and parameters of the cell cycle could not be examined at the single cell
level. It is our experience that Vero cells after a double-thymidine block
start within a few hours to desynchronize. Therefore, the cell-cycle-dependent
expression of Gb3 and GM1 can be reliably correlated only by analyzing single
cells. Interestingly Pudymaitis et al.
(Pudymaitis et al., 1991
)
observed that in verotoxin-resistant Vero cells the decreased toxin binding
was paralleled by a decreased cellular Gb3 content and a strong reduction of
the activity of UDP-galactose:lactosylceramide-
-galactosyltransferase,
which converts lactosylceramide into Gb3
(Kojima et al., 2000
;
Keusch et al., 2000
). As we
found here an inverse relationship between the binding of CTX and that of ST
we considered the possibility that the biosynthetic reactions leading to GM1
and Gb3 respectively might compete for the common precursor lactosylceramide
and that a cell-cycle-dependent change in the activity of
-galactosyl-transferase-activity might be responsible for the observed
changes. However, as shown here by the experiments with the ST-resistant
Vero317-cell line, the cell-cycle-dependent changes in the expression of GM1
on the cell surface occurred in the same way as in normal, ST-sensitive cells.
These data were confirmed by our experiments with PC12 cells.
At the moment we can only speculate on the biochemical mechanism underlying
the cell-cycle-dependent expression of GM1 on one hand and Gb3 on the other.
These mechanisms must act downstream of the synthesis of lactosylceramide as
this metabolite is a common precursor for both types of glycosphingolipids.
Synthesis of Gb3 may then be regulated by the cell-cycle-dependent expression
of the highly specific -galactosyltransferase
(Kojima et al., 2000
;
Keusch et al., 2000
).
The biosynthesis of GM1 may involve several steps. An
UDP-N-acetyl-galactosyl transferase converts lactosylceramide to GA2, and GM3
to GM2, both precursors of the CTX-receptor GM1. It has been reported in a
hybrid rat glioma-neuroblastoma cell line, that the activity of this enzyme
reaches a maximum during S- and G2-phase
(Scheideler et al., 1984).
This is not in line with our observation that the expression of CTX receptors
on the cell surface reaches a minimum during S- and G2-phase.
Another possibility would be a cell-cycle-dependent regulation of
sialyltransferase I, which catalyzes the conversion of lactosylceramide to
GM3, and of GA2 to GA1, both precursors for GM1. The activity of this enzyme
reaches its maximum during telophase and G1 and is elevated in
contact-inhibited cultured cells (Burczak
et al., 1984). Another mechanism has also to be considered, namely
regulation by breakdown. Scheideler et al.
(Scheideler et al., 1984
)
observed, in a synchronized hybrid rat glioma-neuroblastoma cell line, a
strong increase in the activities of N-acetyl-ß-hexosaminidase and of
ß-galactosidase 18-20 hours after release of the cells from a block in
G1. This would fit our observation of a very low binding of CTX in S- and
G2-phase. As the activities of the glycospingolipid-degrading enzymes undergo
a complex regulation by sphingolipid activator proteins (SAP-A to SAP-D and
the GM2 activator) (for a review, see
Fürst and
Sandhoff, 1992
) it seems possible that these regulatory proteins
may also be involved in regulating the net amount of the AB5 receptors and
their expression on the cell surface.
The question is does cell-cycle-dependent expression of Gb3 and GM1
represent an epiphenomenon or does it play a regulatory role in the
progression of the cell cycle. Physiological ligands for Gb3 or GM1 have not
been reported so far. However, effects of glycosphingolipids on growth-related
processes have been described. In bovine aortic endothelial cells GM1 or GM2
inhibit bFGF-induced mitogenesis by interfering with bFGF binding, whereas GM3
is synergistic with bFGF (Slevin et al.,
1999). In T-lymphocytes gangliosides inhibit the interaction of
IL-2 with its plasma membrane receptor, which was explained by a direct
interaction of IL-2 with the gangliosides
(Chu et al., 1993
). In the
fetal cell lines, CH I and CH II gangliosides inhibit
[3H]-thymidine incorporation into DNA
(Icard-Liepkalns et al.,
1982
). In keratinocytes, GM3, but not GM1 or GD1a, stimulates
proliferation (Paller et al.,
1993
), which contrasts with the situation in K562 leukemia cells,
where GM3 inhibits growth, while GM1, GM2 or GD1a have no effect
(Nakamura et al., 1991
). In
lung adenocarcinoma cells, GM3 inhibits the proliferative action of EGF, most
probably by activation of a EGF-receptor-directed phosphotyrosine phosphatase
(Suarez Pestana et al., 1997
).
Inhibition by gangliosides of T-cell proliferation seems also to result from
the activation of a protein phosphatase, in this case resulting in a
diminished phosphorylation of the retinoblastoma protein
(Irani, 1998
). In PC12 cells,
GM1 potentiates the action of NGF by tight binding to the NGF receptor, thus
enhancing its tyrosine kinase activity and dimerization
(Mutoh et al., 1995
). In the
absence of NGF, GM1 did not have any effect. On the other hand, the mere
overexpression of GD3, GD1b and GT1b induced dimerization and a long-lasting
activation of the NGF-receptor TrkA with subsequent activation of the
Ras/MEK/ERK pathway (Fukumoto et al.,
2000
). Under these conditions the sensitivity towards NGF was lost
but could be restored by the addition of GM1
(Mutoh et al., 1995
).
It thus appears that glycosphingolipids are involved in the tuning of complex processes like growth and differentiation, which are tightly linked to cell cycle activity. Further progress will require a knowledge of the physiological ligands for the glycosphingolipid receptors on the plasma membrane.
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Acknowledgments |
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References |
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