CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
LECTIN-INDUCED AGGLUTINATION
11
OF SEPARATED SUBPOPULATIONS OF TERATOCARCINOMA
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Carol Marie Koprowski
June, 1979
The Thesis of Carol Marie Koprowski is approved:
Marvin Cantor
Phillip theeler
Steven B. Oppenhei~, Committee Chairman
California State University, Northridge
ii
ACKNOWLEDGEMENTS
I wish to express my appreciation to the many people
who supported and encouraged me through this work, especially my husband, Ed, and my_ family.
I am also thankful
to Dr. Steven B. Oppenheimer for his guidance and patience,
and to Dr. Marvin Cantor and Dr. Phillip Sheeler for serving as members of my graduate committee.'
iii
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS
LIST OF TABLES •
iii
. . ... . .. ..
LIST OF FIGURES.
v
vi
ABSTRACT • • . • •
vii
I. INTRODUCTION • • • • .
•
1
II. MATERIALS AND METHODS • • .
4
Reagents and Media • •
4
Preparation of Cell Suspensions.
4
Density Gradients • • . • • •
5
Zonal Separation of Cells • •
6
•
..
Cell Counting and Sizing •
Agglutination Assay • • •
Fluorescence Microscopy • •
Zonal Separations • • • • •
Agglutina~ion
.
7
8
10
III. RESULTS . • •
Cell
. . .
7
Assays.
Fluorescence Microscopy.
10
10
13
20
IV. DISCUSSION • •
26
V. BIBLIOGRAPHY • •
iv
LIST OF TABLES
Table
Page
1. Summary of lectin experiments on specific
teratocarcinoma populations. . • • • . • • •
v
14
LIST OF FIGURES
Page
Figure
1. Separation of teratocarcinoma cell populations using the reorienting gradient
zonal rotor • • • • • • • • • •
. ...
LPA agglutination assay. . . . . . .
LTA agglutination assay.
. ...
UEA-1 agglutination assay. .
. .
2. SBA agglutination assay.
3.
4.
5.
vi
...
.
.. . .
.
12
16
17
18
19
ABSTRACT
LECTIN-INDUCED AGGLUTINATION
OF SEPARATED SUBPOPULATIONS OF TERATOCARCINOMA
by
Carol Marie,Koprowski
Master of Science in Biology
A heterogeneous mixture of 129/J mouse teratocarcinoma
cells were separated into two populations of cells on the
basis of cell size and density using centrifugal force.
The teratocarcinoma consistently separated into two populations, "large" cells and "small" cells.
These two popula-
tions of cells were each studied in regards to their surface characteristics by the use of lectin assays and fluorescence microscopy.
tinin (SBA)
1
Lectins used included soybean agglu-
Limulus polyphemus (LPA) , Ulex europus-I agglu-
tinin (UEA-1), Lotus tetragonolobus agglutinin (LTA), and
their fluorescein conjugates.
The results of this study
demonstrate that while both populations may have the ability to bind lectin, only the large cells were agglutinated
vii
- - - - - - - - - - - - - - ------
---~===---~-~~-'-=--"~=--=-=-=---=-----=-=-=-=:---==- ~ =-""-~--=~--
to any appreciable degree.
--
---=:=._~_-::__-,---,:-___:::____·
-
----= ---=------=--=---=---
SBA was the only lectin that
bound to both cell po.pulations and induced agglutination in
only the large cell population.
LPA, LTA, and UEA-1 did
not induce appreciable agglutination in either population.
Fluorescence microscopy revealed that LPA bound only to the
small cell population, while LTA and UEA-1 bound to neither
cell population.
The large cell population resembles mal-
ignant or transformed cells in regards to lectin-induced
agglutinability characteristics, while the small cells have
agglutinability characteristics typical of benign cells.
The disparity observed in agglutination of the different
populations may be due to the involvement of cytoskeletal
rearrangements.
viii
-~--
----------:--
INTRODUCTION
The,study of cancer involves a comparison of malignant
or transformed cells and normal or benign cells.
There are
two major differences between malignant and benign cellsi
(1) A loss of "contact inhibition" and
(2) A loss of cell-cell adhesiveness.
Contact inhibition is the inhibition of cell motility and
mitotic activity when cells grown in tissue culture come
into contact with each other (DeRobertis, 1975).
With a
loss of contact inhibition, malignant cells resume continuous uninhibited mitotic cell division.
When cells become
less "sticky" they may break free from the remaining cellular. mass and invade other parts of the body.
One method for studying malignant cells involves the
use of lectin-macromolecules extracted from such sources as
plant seeds, roots, bark, leaves, and vertebrates and invertebrates (Sharon and Lis, 1972).
Lectins are operation-
ally defined as polyvalent, carbohydrate-binding proteins
(Barondes, 1977).
They possess several important proper-
ties that make them useful in biological applications,
among which are the abilities to bind to sugars, to induce
1
2
mitosis in lymphocytes, and to agglutinate malignant cells
(Sharon and Lis, 1972).
Lectins are especially useful and
valuable in studying cancer cells because they can distinquish differences in the surfaces of cells and can agglutinate transformed, trypsinized, and embryonic cells.
Lee-
tins bind to normal cells but do not induce agglutination
(Aub
~
sl, 1963; Ambrose
Pollack~
~'
1969; Inbar
~
al, 1961; Burger
~ ~~
~.
al, 1967;
1969; Inbar and Sachs,
--
--
1969; Burger, 1969; Eckhart et al, 1971; Rabinowitz et al,
Cline~ ~,
1970;
1971; Ozanne
~ ~'
1971; Roberson et al1
19 75) .
The nature of the interaction of cell surface receptor
sites to lectins remains unclear.
the involvement of a
wrapper around cells.
11
Burger (1969) proposed
Cryptic site, 11 a protein coat or
The lack of this wrapper in trans-
formed cells allowed the exposed receptor sites to bind
lectin.
Other investigators, however, found that normal
cells have the ability to bind equivalent quantities of
lectin as do transformed cells
(Mallucci, 1971; Cline and
Livingston, 1971; Ozanne and Sambrook, 1971).
Still other
studies suggest that transformation is accompanied by cytoskeletal rearrangements that affect the mobility of the receptor sites and thus alter the cells' ability to be agglutinated by lectins (Nicolson, 1971; Nicolson, 1972).
In
any case, lectin assays remain a valuable tool in the study
of malignant and benign cells.
3
To date, lectin assays of teratocarcinoma cells have
utilized heterogeneous mixtures of cells.
Teratocarcinoma
is known to consist of primitive malignant stem cells
(known as an embryonal carcinoma, which resembles early embryonic cells) and the benign, more differentiated cell
types derived from these primitive stem cells.
The present
study involves the separation of a heterogeneous population
of cells on the basis of cell size and density and the
testing of the resulting populations of cells for the abili·
ty to bind and be agglutinated by lectins.
The results in-
dicate that a heterogeneous mixture of cells may be consist
ently separated into two populations, "large" and "small".
On the basis of lectin assays, the large cell population
exhibits characteristics typical of maligni3.nt or transformed cells, while the small cell population exhibits
those properties characteristic of normal or adult cells.
MATERIALS AND METHODS
Reagents and Media
Lectins and their FITC-derivatives:
Limulus polyphe-
~
(LPA), Ulex europus-type I
~
(LTA), soybean agglutinin (SBA), Ficoll (type 400),
(UEA-I), Lotus tetragonolo-
sucrose, N-acetyl-D-galactosamine,
~-L-fucose,
fetuin,
N-2-hydroxyethypiperazine-N'-2-ethanesulfonic acid (Hepes
buffer), Deoxyribonuclease I
Sigma Chemical Co.
(DNAse) were obtained from
(St. Louis, Missouri) and an additional
complete set of lectins were obtained from E.Y. Labs (San
Mateo, CA.).
cated sources:
Other materials were obtained from the indiIsoton, Coulter Electronics, Hialeah, Flo.;
trypan blue, MCB Chemicals.
Glucose-free Hepes buffered
Hanks' solution (GFHH) was prepared according to Hanks
(Hanks and Wallace, 1949) with the following modifications:
glucose and phenol red were omitted and 0.01
!:1.
H,epes was
included.
Preparation of Cell Suspensions
The 129/J ascites teratocarcinoma was obtained from
Dr. Leroy Stevens of the Jackson Laboratory.
4
The cell line
--
---
--
-----
----------------------------------~--------- -----------------------------------~--
- - -
5
used was OTT 6050 which grows as single undifferentiated
embryonal cells; therefore no treatment with proteolytic
enzymes was needed to obtain single cells for agglutination
studies.
Studies involving the use of lectins are more re-
liable if the need for proteolytic enzymes to obtain single
cells can be eliminated.
Such enzyme treatments have been
shown to alter the nature of the cell surface and its reaction to lectins (Childress .§j; al, 1979).
Cells used in
this study, therefore, are unaltered and possess undisturbed surfaces.
The cells were maintained in young male 129/J mice
by intraperitoneal passage.
Mice were sacrificed by cer-
vical dislocation when abdominal bloating occurred (5-15
days).
The peritoneal contents of the mice were removed
and the cells diluted 1:2 in cold GFHH (pH 7.4).
suspension was gently centrifuged in
The cells
15 ml conical cen-
trifuge tubes for 5 min at 186 x g in an I.E.C. clinical
centrifuge.
The supernatant was removed and the procedure
repeated twice to complete the washing.
A final cell sus-
pension was made in GFHH, which had been adjusted to 1%
w/w in Ficoll.
Density Gradients
Density gradient limits were prepared using Ficoll 400
as the gradient solute in GFHH.
A 50 ml 50% wjw sucrose
cushion (underlay) was prepared using sucrose in water.
a typical run, 50 ml cushion, 600 ml exponential density
In
---
----~---
---------------------- ----------------- --------------------------------
--------------
---------
--- - - - - - - - - - - - -
6
gradient, and 600 ml linear gradient were used.
The expo-
nential gradient was prepared using 100 ml 20% w/w Ficoll
in the mixing chamber and a reservoir containing 600 ml 8%
w/w Ficoll.
The linear density gradient was prepared using
300 ml 8% w/w Ficoll for the dense limit and 300 ml 4% w/w
Ficoll for the light limit.
ge~erated
Both density gradients were
using a DuPont/Sorvall GF-2 gradient maker with
delivery and removal using a peristaltic pump.
Zonal Separations of Cells
Zonal separations of cells were obtained using a DuPont/Sorvall TZ-28 Reorienting Gradient Zonal Rotor and a
DuPont/Sorvall OTD-50 oil turbine drive ultra-centrifuge.
The 50 ml cushion was loaded at 1340 rpm and was followed
by the exponential and linear density gradients.
gradients were loaded dense end first.
Both
A centrifugal force
of 90 x g was present at the gradient surface.
At this
time, the centrifuge was switched to the "reograd" mode and
the sample (25 ml containing 3.0 x 107 cells) was sprayed
into the rotor through the annular loading ring.
The rotor
decelerated to rest over a 16 min interval, which provided
a total applied force of 170 g-min.
A stroboscopic lamp
was used during deceleration to assure consistency in rotor
speeds during successive runs.
The OTD-50 was found to
provide reproducible, smooth, and gradual rotor decelerations.
The density gradient containing the separated cells
was removed from the rotor and collected as a series of
7
50 ml fractions.
Cell Counting and Sizing
The numbers and sizes of cells in both cell suspensions loaded onto the gradient and the separated cells which
were later collected were determined using a Model B Coulter
Counter equipped with a Model J size distribution plotter.
Any fractions requiring preliminary dilution were adjusted
with GFHH prior to counting and sizing.
Agglutination Assay
A reliable and quantitative method for ascertaining
cell agglutination has been previously described by Oppenheimer and Odenkrantz (1975).
This method involves count-
ing the disappearance of single cells into aggregates of a
rotating cell suspension using an electronic particle counter.
Both large and small single cells were counted to the
exclusion of cell aggregates.
The collected fractions containing either large or
small cells were pooled and the pooled cells then centrifuged at 654 x g for 5 min in a DuPont/Sorvall GSA rotor.
The cell pellets were washed twice and suspended in GFHH at
a concentration of 2.4 x 10 4 cells/ml.
It was only on
these separated populations of cells that agglutinatiqn
assays were performed.
Lectin concentrations were prepared to yield a final
reaction concentration of 250, 125, 62.5, 32, 16, 8, 4, 2,
--- - - - - - - - - - - - -
----------------------------
- ----
----
--------------
~---~-
--
8
1 and 0.5 ug lectin/ml cell suspension.
assay consisted of taking 100
~1
The agglutination
of cell suspension and
100 ul of lectin solution and placing the mixture on a gyratory shaker at 37°C for 20 min.
For the control, 100 ul of
GFHH was added in place of the 100 pl of lectin.
After the
incubation period, the cells were then assayed for the presence of single cells (as described above).
The percentage
of cells agglutinated was computed by comparing the cells
present in the cell/lectin mixtures with the single cells
present in the (no lectin) controls.
Viability tests were
performed using the trypan blue dye exclusion technique
(Roth, McQuire, and Roseman, 1971).
Fluorescence Microscopy
The separated populations of cells were incubated with
the fluorescein isothiocynate-lectin conjugate (FITC-lecti~
to visually confirm what was observed in the agglutination
assay.
100
A
~olume
of 100 pl of cell suspension was added to
pl of 50 pg/ml FITC-lectin and placed on a gyratory
shaker for 20 min at 37°C.
The cells were washed twice and
then fixed in 4% formaldehyde for 20 min.
The cells were
given a final wash and then examined for fluores.cence activity and cell agglutination using a Zeiss Fluorescence
microscope with incident illumination.
The cells were ex-
amined on two separate days to visually confirm agglutination.
By the same procedure, hapten inhibition studies were
9
performed with the following modification.
The cells were
preincubated for 5 min with the appropriate inhibitory
sugar (see Table 1) at a concentration of 0.1 M sugar
prior to the addition of FITC-lectin.
e .
RESULTS
Zonal Separations
The mouse teratocarcinoma used in this study consists
of a heterogeneous mixture of cell sizes.
Using the tech-
nique of zonal centrifugation this mixture could be consistently separated into two populations of cells which
were labelled "large" cells (approximately 20-30 p. in diameter) and "small" cells (approximately 7-11 Jl in diameter).
Figure 1 shows the results of a typical separation run presented in the form of a "perspective plot," which clearly
shows the existence of two separate cell populations.
Usin~
this separation technique it was possible to obtain
a sufficient quantity of cells which cculd then be tested
further in regards to cell surface characteristics.
Cell Agglutination Assays
Each assay utilized lectins from two different sources.
Similar results were obtained with different lectins having
similar sugar specificities.
Hapten inhibition experiments with the correct sugars
indicated that the purified lectins used in these experiments agglutinated cells with appropriate sugar specifici-
10
11
FIGURE 1:
Separation of teratocarcinoma cell pop-
ulations using the reorienting gradient zonal rotor.
3 x
10 7 cells were placed on a density gradient varying linearly from 2-8% w/w Ficoll-GFHH over the first 600 ml and exponentially from 8-20% wjw Ficoll-GFHH over the second 600
ml.
The density gradient was removed from the rotor dense
end first.
The prospective plot was generated using the
TRID program from the Cal Comp Plotter software library, on
a Control Data Corporation 3170 computer which was interlaced to a Cal Comp computer.
12
1.0"
F
FIGURE 1
....I
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0
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FRACTION NUMBER
25
13
ties (Table 1).
In all experiments, a decrease in the num-
ber of single cells over time was a result of viable agglutination and not cell lysis.
Cell viability corresponded
to an average value of 90% (+ 5%) using the trypan blue dye
exclusion technique.
Table 1 and Figures 2-5 summarize the agglutination
versus concentration data for each lectin used.
SBA was
the only lectin that induced appreciable agglutination of
the large cell population but none in the small cells.
LTA,
UEA-1, and LPA caused no agglutination in either population.
Similar results using other lectins were obtained by Childress
~
~
(1979)
and are summarized in Table 1.
Fluorescence Microscopl
Binding of the lectin,visualized with fluorescence
microscopy, did not necessarily yield agglutination of the
- cells.
As can be seen in the results, FITC-SBA bound to
both subpopulations of the cells, but induced agglutination
only in the "large" cell population.
The FITC-conjugates
of LTA and UEA-1 bound to neither cell population and correspondingly did not agglutinate the cells to any appreciable degree.
Although LPA bound to the "small" .cell popu-
lation, it caused no agglutination there.
Appropriate hap-
ten inhibitors prevented the binding of lectin to both cell
types (see Table 1) •
TABLE 1
SUMMARY OF LECTIN EXPERIMENTS ON SPECIFIC TERATOCARCINOMA POPULATIONS
LECTIN
BINDING
Lg
Sm
AGGLUTINATION
Lg
Sm
BINDING IN PRESENCE OF HAPTEN
Sugar Spec.
Lg
Sm
REFERENCE
Con A
+
+
+
-
D(
-D-Man
-
-
Childress
RCA-1
+
+
+
-
,8 -D-Gal
-
-
Childress .§.:!;; al (1979)
SBA
+
+
+
-
-
Lis t l al (19 70)
WGA
+
+ slight slight
-
-
Childress~
PNA
-
+
-
-
D-Gal
-
-
Childress
LPA
-
+
-
-
Sialic Acid
-
-
Roche et al (1975)
LTA
-
-
-
-
o(
-L-Fucose
-
-
Etzler
UEA-1
-
-
-
-
D(
-L-Fucose
-
-
Matsumoto .§.t al ( 19 72)
N-D-GalNH 2
N-D-GlcNH
2
n
tl
n
~
£1
(1979)
{1979)
al {1979)
al (1974)
I-'
,j>o
15
FIGURES 2-5:
agglutination.
Effect of lectin concentration on cell
The concentrations of lectins range from
0.5 to 250 ug/ml • .-.designates large cell assays, while
&---4
designates small cell assays.
16
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DISCUSSION
The results in this study demonstrate that it is possible to obtain two populations of cells from a heterogeneous population of teratocarcinoma cells:
population and a "small" cell population.
a "large" cell
Both populations
can bind lectin, but only the large cells are agglutinated
by lectin to any significant degree.
These results suggest
that the large cell population resembles transformed or
certain embryonic cells while the small cell population resembles normal or adult cells with respect to agglutination.
Sharon and Lis (1972) demonstrated that if both tumor
(transformed) and normal cells were incubated with lectin
(specifically SBA) only the tumor cells were agglutinated.
It was also shown that both tumor cells and embryonic cells
were similar in their response to lectins (Roberson and
Oppenheimer, 1975).
Further work done by Mallucci (1971)
using immunofluorescence and Kline and Livingston (1971)
and Ozane and Sambrook (1971), using radioactively labelled
lectin, showed that both normal and transformed cells were
capable of binding the same amount of lectin.
This indi-
cated that some altered surface membrane property of trans-
20
21
formed cells allowed them to agglutinate in the presence of
lectin.
Similar results were obtained using embryonic
cells and normal cells by
Krach~
al.
(1974).
Therefore,
while both cell types have the ability to bind lectin, only
transformed or embryonic cells may be agglutinated by leetin.
It has been known for some time, however,
b~at
the
surfaces of normal cells and those of malignant or transformed cells react differently with lectins.
Various ex-
planations and models have been proposed in an attempt to
explain this difference.
The most promising model presen-
ted so far suggests that transformation is accompanied by
cytoskeletal rearrangements which render the receptor-sites
more mobile (Nicolson, 1971 and 1972).
Nicolson proposed
that there was a specific topographical distribution of
Con A receptor sites found on the surface of trypsinized or
transformed cells which accounts for their agglutinability.
This difference in distribution (lectin mediated) which appears as clustering of Con A receptor sites may be responsible for the subsequent agglutination of these cells.
Earlier studies have indicated that the cell surface
macromolecules, which are capable of two-dimensional movement in the plane of the membrane (Frye and Edidin, 1973),
may have their mobility altered upon the binding of lectins.
Rosenblith (1973)found that only transformed or trypsinized
cells were capable of Con A receptor clustering and that
22
normal cells were not capable of this.
DePetris (1973)
tested this hypothesis further and found that although the
clustering at lectin receptor sites was necessary, it was
not sufficient to account for the observed difference in
agglutination.
One possible explanation to account for this difference in agglutination could be the hypothesis proposed by
Edelson, Yahara, and Wang (1973) and again by Yahara and
Edelman (1973).
They suggest that receptor sites occur in
two states, mobile or attached, depending upon the state of
a colchicine-binding protein system.
This protein system,
the identity of which is not known, can be altered by colchicine or other related drugs and may be related to actin
like proteins and microtubules.
In the mobile state the
receptors are attached to a colchicine-binding protein system which is dissociated from itself, while in an attached
state the receptors are attached to a colchicine-binding
protein system which is intact.
Based on their hypothesis,
binding of lectin alters ·the mobility of the colchicine
binding protein system which in turn alters the cell membrane.
This hypothesis is based on the observation that
the plasma membrane has a fluid nature which enables receptors to move through a two-dimensional plane (DePetris,
1972).
Furthermore, Nicolson and Yanagimachi (1974) were
able to demonstrate the existance of localized restraints
on the mobilities of lectin receptors in specific regions
23
on plasma membranes of rabbit spermatozoa, and postulated
the possible involvement of transmembrane restraints to
maintain this segregation.
Yahara and Edelman (1972) found that the binding of
Con A led to a change in the cell surface or cell membrane
that resulted in the alteration in the anchorage lymphocyte
immunoglobulin receptors.
Evidence for the involvement of
the cytoskeletal (microtubule) system was demonstrated by
Berlin and Ukena (1972) and Yin, Ukena, and Berlin (1972)
by studying the effects of colchicine, colcemid, and vinblastine on the agglutination of transformed cells.
Zagyansky and Edidin (1976) proposed that the addition
of lectin (Con A specifically) to transformed and normal
cells alters the clustering or anchoring of receptor molecules so as to greatly inhibit their diffusion in the plane
of the plasma membrane.
They proposed the following se-
quence to account for the binding of lectin:
(a) Transformed cells, bearing highly mobile receptors,
bind lectin and rapidly reform the receptors into aggregates.
This aggregation of receptors triggers their immob-
ilization by anchoring into a submembrane cytoplasmic system.
(b) Non-transformed cells (contact inhibited cells)
bear aggregates of receptors, roughly an order of magnitude
larger than those found on transformed cells.
gates are found to have a
receptor~to-receptor
These aggreor a recep-
--~~---~-----~
24
tor-to-cytoplasmic anchor.
Upon the addition of
lecti~
these pre-existing aggregates come together anchoring them
again.
The binding of lectin and subsequent restriction of mobility is a two-step process dependent upon the initial diffusionable mobility of the receptors
(thereby making this
mobility higher in transformed cells).
Earlier work done by
Loor, Forni, Pernis (1972) using lymphocytes supports this
idea.
The present results are not inconsistent with the
hypothesis of Nicolson (1971).
Testicular teratocarcinoma are tumors of 129/J mice
consisting of primitive malignant stem cells (known as embryonal carcinoma cells and resembling early embryonic
cells) and differentiated cell types arising from these stem
cells (Stevens, 1967).
Stevens and Little (1954) found that
testicular teratocarcinomas are true tumors possessing the
ability of progressive growth and composed of embryonic and
adult tissues not normally found in the testis.
Later work
by Pierce and Beals (1964) found that the ultrastructure of
primordial germ cells strongly resembled that of embryonal
carcinoma cells and proposed that the primordial germ cell
was the cell of origin of teratocarcinoma.
Other studies
of Pierce, Stevens, and Nakane (1967) and Stevens (1967)
lent further support to this hypothesis.
In order to test
the multipotentiality of embryonal carcinoma cells, Klein,
Smith, and Pierce (1964) performed 1700 single cell grafts
25
of which 44 clonal lines resulted.
Studies of these lines
revealed that 43 lines were teratocarcinomas composed of as
many as 14 well differentiated somatic tissues along with
embryonal carcinoma cells.
It can be said then, with rea-
sonable certainty, that the embryonal carcinoma cell, derived from primordial germ cells, is the multipotential
stem cells of testicular teratocarcinoma which has the ability to differentiate into benign somatic tissue cells.
The
present results indicate that the large cell population
possess the surface characteristics typical of malignant
stem cells or embryonal carcinoma cells.
The small cell
population possess the surface characteristics typical of
benign cells.
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