Download the surface morphology and the cell cycle of mastocytoma

AJEBAK 54 (Pt. 2) 137-147 (1976)
THE SURFACE MORPHOLOGY AND THE
CELL CYCLE OF MASTOCYTOMA TARCET CELLS:
NO APPARENT EFFECT ON CELL-MEDIATED KILLING
by L. M. CHING, J. B. GAVIN*, I. MARBROOK AND M . SKINNER
(From the Departments of Cell Biology and Pathology",
University of Auckland, New Zealand.)
{Accepted for publication Febrtiarif 19, 1976.)
Summary. Mastocytoma cells (P-185) have been separated by velocity sedimentation into fractions which were highly enriched for eells at discrete stages
of the cell cycle. By scamiing electron microscopy it was shown that the surface
morphology of the majority of cells in each fraction was characteristic of that
fraction. No diiference could be detected between i.solated fractions and
unfractionated cells in their ability to be lysed by cytotoxic lymphocytes.
INTRODUGTION.
The main stages of a cell cycle are defined in relation to the time of DNA
synthesis and mitotic division (Howard and Pole, 1953). Many other characteristics have been shown to vary with the stages of division. Gell cycledependent changes in the external surface are of particular interest in stitdies
on cell interactions. These changes include gross alterations in the structure of
the membrane (Scott and Garter, 1971), fluctuations in membrane potential
(Sachs, Stambrook and Ebert, 1974). surface charge (Brent and Forrester,
1967) and variations in siuface morphology (Porter, Prescott and Frye, 1973).
As immune reactions against cells involve antigens on the surface of the
target cell, the variation of antigenic sites at different stages of the cell cycle
are potentiaHy important in immnne cytolysis. Gyclic variations of the histocompatihility H2 antigen have been observed with cultured mastocytoma cells
(Pasternak, VValmsley and Thomas, 1971) and in virus transformed mouse
lymphoma cells (Gikes and Friberg, 1971). The H2 antigens were maximally
expressed in early G] and minimally in S-phase. The sensitivity of cells to
antibody-mediated cytolysis has been examined throughout the cell cycle
(Shipley, 1971; Lerner, Oldstone and Gooper, 1971) but cell-mediated cytolysis
has not been studied in such detail.
138
L. CHING. |. GAVIN, f. MARBROOK AND M. SKINNER
This paper describes an investigation of the surface morphology and susceptibility to cell-mediated immune lysis of mastocytoma cells at various stages
of the cell cycle. The technicjiie of velocity sedimentation {Miller and Phillips,
1969) was used to obtain fractions which were higlily enriched for cells at a
given stage of the cell cycle.
MATERIALS AND METHODS.
Cell line.
The mastocytoma P185 was u.scd throughoiil. The line was inaintainecl a.s an ascitic
tiiiiiour in (DBA/2 x C.,H) F, Iiybnd mice by transferring lO-"' cells every 8-10 days. Five
clays after cell transfer, proliferating mastocytoma cell popiilation.s were removed from the
peritoneal cavity in 5 ml of pho.sphate budered saline (PBS) (pH 6 5 017M NaCl
00034M KCI, OOIM NaoHPO,. OOIM KfL-PO^).
Pulse labelling of cells.
Cells vi'ere labelled in vivo hy intra peritoneal injection of 2-5 nCi {-'Hl-thyniidine
(specific activity 240 mCi/inmole) or 0-5 ^Ci ('*C)-thymidine (.specific activity 59 inCi/
niinole).
Velocity sedimentation.
The general methods described by Miller and Phillips (1969) were used with some
modifications. All sedimentations were conducted at 4° in a cylindrical glass chamber
(10-25 cm diameter) with a conical base connected by Tygon tubing to a peristaltic pump.
Fifteen ml PBS, followed by 3 x 10" cells in 10 ml of 4% calf serum in PBS, then 400 ml
of gradient (7-25? calf serum in PBS) were pumped tlirough the base into the c'hamber. The
cells were allowed to sediment for 3 li at unit gravit>, then 10 ml fractions were collected
with an automatic fraction collector (30 fractions/h). The ability of the cells to form colonies
in agar wa.s used to indicate that there was no loss of viability during fractionation.
Measurement of radioactivity.
Cells were collected on glass fibre filters, which were then washed with distilled water
to remove calf serum and v\ith 5% trichloroacetic acid to remove acid-soluble radioactivity.
They were then dried and plac-ed in vials with 5 ml scintillation fluid (7-5 g 2,5-diphenyIoxazole and 0-25 g 1,4-bis [2-(5-phenyioxazolyl )]-benzene to 2-5 litres of toluene). The
radioucti\ity wa.s measured by liquid seintillatinn counting.
Scatminfi electron microscopy ( SEM).
Cells were fixed at room temperature in 30 niin changes of 0-5%, I I , 2% pho.sphate
bufi^ercd glutavaldehyde then washed and dehydrated iu increasing concentrations of tertiary
butanol (Wheeler, Seelye and Gavin. 1975). Drops of the.se suspensions were placed on
aluminium stubs, dried under vacuum, coated with carbon, then gold-palladium, and examined
in the .scanning electron microscope (Stereoscan 2A, Cambridge Scientific Instruments Ltd..
England).
Cell-mediated imj}titne killing.
Cytotoxic lymphocytes, sensitized against DBA/2 histocoinpatibility antigens, were
obtained from spleens of (DBA/2 x C-jH) Fj hybrid mice which had been irradiated
(800 rads) and injected 5 days previously with 10« nucleated spleen cells from CBA mice.
Spleen cell suspensions were prepared by teasing out the .spleen into medium (RPMI 1640
GIBCO). The cells were irrigated through a 20 gauge needle and clumps were allowed to
settle into ealf serum.
GELL GYGLE STAGES OF TARGET GELLS
139
Cell-mediated lysis was measured by the '>' Ci-chrnmate ielea.se assay (Biunner ct al..
1970). Four .\ 10" nia.stocyt()ma cells; were labelled with 20-30 (iCi ^''Cr-chromate in 0-5 ml
balanced salt solution for 30 min at 37°, then washed thoroughly to remove unbound
chromate. Ten thousand labelled cells were added to the appropriate dilution of spleen cells
in a total volume of 0-5 ml medium (RPMI 1640). Cells were centrifujied at 90 g and.
after a 4 h incubation at 37° in an atmo.sphere of 5% CO^ in air, 1-5 inl of PBS \va.s added
to each tube. The cells were thoroughly dispersed and then scdimentet! by centrifugation.
One ml of tlu" supematant was removed and the radioactivity coimted in a Cannna Counter.
Results were expressed as:
counts
100
pereent chromium released =
=
counts in
in supematant
supematant
X
totat counts meorporated into the target cells
RadiomttogTaphy.
Cells suspended in calf serum were smeared on to mieroscope slides, then fixed in
absolute ethanol (10 min) and 2% acetic acid (5 min). The slides were washed in distilled
water, dried and coated with Kodak NTB-2 liquid emulsion. After 5 days at 4° the radioautographs were developed and the slides were stained with 5« Ciemsa stain (5 min), washed
in phosphate buller. dried and scored for "labelled" cells.
RESULTS.
The separation of asynchronous nuistoctjtoma cells into fractions
containing cells at discrete stages of division.
Ma.stocytoma cells growing as an ascitic tnnionr were taken in the middle
of the exponential phase of growth and fractionated according to thek cell
volume, using velocity sedimentation. To verify that the cell fractions represented
discrete stages of the cell cycle, the sedimentation rate of a stib-population of
ceils, labelled with ''H-thymidinc, was followed thronghont one growth cvcle.
A pnlse of ^H-Tdr was injected intraperitoneally into a series of mice and
the tumour cells harvested at various times thereafter. The total amount of
•'H-Tdr was incorporated into acid-precipitable radioactivity in less than 10 min
( < 0 0 2 of generation time), Immediately before removing the ttnnour cells
from the peritoneal cavity, the population received a second pulse of '"Gthymidine to label the cells in S-phase at the time of removal. This gave a
reference population so that both the absolute and the relative seditnentation
rates of cells could be estimated.
The sedimentation profiles of radioactive cells which had grown for various
lengths of time are shown in Fig. 1. When tho tritium- and '^carbon-thyniidine
were added simultaneously, the radioactive population sedimented at a mean
rate of 8-5 mm/h. As the interval between the incorporation of the two radioactive isotopes increased, the tritium-labelled sub-population sedimented at a
higher rate than the standard S-phase cells. After 4 h, the •'H-labelled cells
sedimented as two populations, indicating tbat some of the cells had passed
through G:- and mitosis and had reached G,. The cells in G, sedimented at a
rate of 6-3 mm/h. B\ 7 h, all the •'•H-labelled cells had divided and they
140
L. GHING, |. GAVIN, ]. MARBROOK AND M . SKINNER
SEDIMENTATION RATE (mm per h)
4 5
4-5
6 5 8 5 IO'5 12 5
6 5
85105135
f
lOO
5 h
it
A
S^
.''.J
100
A
\
I h
<3
6 h
A ^
a \
\
Q'
»_(»<»
100
2 h
7 ti
50
z
/
\
-..*
k**
2
d
100
3 h
O
8 h
o
50
J/
100
50
A
iJ V
.//
/•'
I
J
9 h
\
Fig. 1. Sedimentation profiles nf nulioacti^-cly-lahelk'd nuvstocytoina cells troni mice which
were injected with -'H-Tdr. From zero to 9 h after the ''H-Tdr injection, the mice were
injected with '^C-Tdr. The cells were harvested 15 min later and fractionated. The radioactivity in each fraction is expressed as a percent of the maximum.
A ~ A ''H radioactivity. Q--Q '''C radioacti\'fty.
CELL GYGLE STAGES OF TARGET GELLS
141
appeared as a single peak of radioactivity in the plot of sedimentation rates.
The cells in Gi continued to grow until they had moved to the original
sedimentation position of 8 5 mm/h at 9 h. The total generation time was
therefore 9 h.
To ensure that the radioactivity in each fraction reflected the distribution
of radioactive cells, the sedimentation profile of radioactive cells was assayed
using radioautography. The results in Fig. 2 indicate that there was a close
correspondence between the amount of radioactivity in each fraction and the
nnniber of labelled cells.
100
-
Z
c
£
o
IU
z
50
o
ae
Q.
Io
o
6
10
14
le
22
FRACTION NUMBER
Fig. 2. A comparison of the number of "labelled" cells and the amount of radioactivity in
eaeh fraction. Ma.stocytoma cells were putse-lahelled with '^H-Tdr. har%este(l :md fractionated.
An aliquot of each fraction was remo\ed for liquid scintillation counting, and the remainder
of the fraction was prepared for riidioautojjraphy. The number of labelled cells detected by
radioautography and the radioactivity in each fraction are expressed as a percent of the
maximum. 0—0 ''H radioactivity. A--A labelled cells.
Calculation of the duration of stages of the cell ctjcle.
The data shown in Fig. 1 were analysed to calculate the length of each
stage of the cell cycle. The area of the peak of radioactivity in cells at the Gi
stage was expressed as a fraction of the total ''H-thymidine in the mastocytoma
poptilation. In Fig. 3 the fraction of radioactivity in Gi is plotted against time.
Between 2-5 and 3 h after S-phase cells were labelled, radioactivity started
moving into the fractions containing Gi cells, indicating that these cells retjuire
approx. 2 75 h to pass through the G^ and M stages. All radioactive cells had
142
L. GHING, J. GAVIN, ]. MABBROOK
.AND M . S K I N N E B
divided after 7 25 h (Fig. 2). This wt)tikl be the length of time required for cells
labelled in early S to progress through S, G^ and mitosis. Thus, the 9 h cell
cycle time of mastocytoma cells ct)nsists of an S-phase of 4-5 h, Gi phase of
1-75 h and combined G^ and mitotic phase of 2-75 h.
The surface inoTphologtj of celh at different stages of cell cycle.
The majority of cells from three fractions, which were known to be highly
enriched for cells at particular stages of the cell cycle, showed consistent and
distinct topography, although each contained a small proportion of atypical and
intermediate forms.
Fraction 7. (Sed. rate 5-7-6-9 mm/h.)
Gells from this fraction, predominantl\- cells at the G, stage, were generally
spherical in shape and had a slightly roughened surface with short bulbous or
"blt'b-like" protrusions.
I 0
•
•
—
•
0-8
I
0-6
Q
0-4
<
P^
0 2
J
I
I
L
TIME (h)
Fig. 3. The axDpearance of radioacti\ity in G, tells following a "pulse label" of the cells with
••'H-thyniidine. The amount of radioactivity in cells whicb have divided (G|) is expressed as
a fraction of the total ''H-thyniidine incorporated. This fraction is plotted against the time
after addition of isotope. The data is derived from the results of Fig. 1.
Fraction 11. (Sed. rate 7-8-9 2 nim/h.)
This fraction, highly enriched for cells in mid S-phase, contained many
rounded cells with sinut)us, ribbon-like surface projections giving tliein a
"ruffled" appearance.
CELL CYCLE STAGES OF TARGET GELLS
143
u c
ja —
c n
O «
144
L. GHING, |. GAVIN, ]. MARBROOK AND M. SKINNEB
Fraction III. (Sed. rate > 9 2 mm/h.)
Although these cells were predominantly Go or mitotic cells, the data in
Fig. 1 indicated that a very small nnmber of S-phase cells were present. Most
of the cells in this fraction had a few small blebs and many long fine microvilli
extending from their surface. A few cells were considerably larger than the rest
and had a relatively smooth surface.
Plate 1 summarizes the relationship between these appearances, the sedimentation rates and the phases of the cell cycle.
Sub-populations of mastoctjtoma eells as target cells.
The stisceptibility of mastocytoma cells to cell-mediated lysis was investigated using the -^'Gr-chromate release method of Brunner et al. (1970). Gells
from three fractions, described above, were labelled with •'"Gr-chromate. Eacb
fraction was found to be labelled with '"Gr-chronmte to the same extent as the
xtnfractionated cells. Tbe lysis of cells was carried out over a range of targetIymphoid cell ratios and the results are expressed in Fig. 4. The extent of lysis
of the individual fractions was indistinguishable frotn the degree of lysis of
unfractionated cells. The rate of lysis of all samples was identical (unpublished
data).
"
.
bO
o
50
40
LU
U
30
20
10
100
SPLEEN:TARSET CELL RATIO
Fig. 4. Cell-mediated immune lysis of mastocytoma cells at dilteient stages of the cell cycle
by .sensitized spleen cells. Alitiuots from each .sample were incubated with graded numbers of
spleen celis and the •'•>'Cr released from dauiiifjed cells after 4 h was measured. Eac-h point
represents the mean of duplicate assays.
O - O Gi- 0 - 0 S. A - A G., ^ M. • - • Non-fractionated ct-Ils.
CELL CYCLE STAGES OF TARGET CELLS
145
DISCUSSION.
By investigating the progress of mastocytoma cells through the cell cycle,
it has been possible to isolate fractions highly enriched for cells at particular
stages for morphological and target cell studies. Ovu- techni(iue of following a
pulse-labelled population was based on the assumption that S-phase cells incorporate radioactive thymidine into acid-precipitable material at a similar rate
clurinjT the whole S-phase. Two lines of evidence indicate tbat this assumption
is valid. First, the sedimentation profile of cells which may be scored as
"labelled" by radioautography is identical to the profile of radioactivity which
was measured as total radioactivity per fraction (Fig. 2). The amount of radioactivity was therefore proportional to tbe number of labelled cells. Second, the
radioactive cells behaved as a single population on fractionation. Miller and
Pbillips (1969) have discussed the homogeneity of populations in their definition
of intrinsic resolutions ^ (where SS is the width at half the height of the peak
and S is the mean sedimentation position of the population from the origin).
For a single homogeneous population, the intrinsic resolution limit has been
reported as varying from 0-lS (Miller and Phillips, 1969) to 0-28 (Williams and
Moore, 1973). The mean resolution of the total labelled sub-population in the
profiles shown in Fig. 1 was 0-28. When the labelled cells sedimented as two
sub-populations, those which had pas.sed mitosis and those which had yet to
divide (Fig. 1, 5 h), the intrinsic resolution was 0-25 and 0-18 respectively.
These values are in agreement with those of other workers and indicate that
the -'H-labelled cells were homogeneous with respect to cell volume. Thus, the
experiment described in Fig. 1 was essentially a means of following the change
of volume of a synchronous sub-population of mastocytoma cells.
The valnes we have obtained for the duration of the mastocytoma cell
cycle from velocity sedimentation analysis are in good agreement witb the valnes
obtained for cultured mastocytoma cells by Schindler et al. (1970) in which
they used separate technicjues to determine the length of each phase. Longer
values for the mastocytoma cell cycle were reported by Bergeron, Walmsley and
Pasternak (1970) using a method which required colcemid to block cells in
mitosis. However, the presence of colcemid bas been shown to slow down the
progress of the cells (Williams aud Carpentieri, 1967).
Data on the sedimentation rate of cells allowed the size of cells in individual
fractions to be correlated with their stage in the cell cycle and showed tbat
cells sedimeuting at 6-3, 8-5 and 9 9 mm/h were highly enriched for Ci, S,
G2 + M cells respectively.
Scanning electron microscopy demonstrated that mastocytoma cells show a
wide variety of surface specialisations whicb are closely related to the stage in
the cell cycle. The order of changes we propose (Plate 1) was based on the
remarkable consistency with which tbe features were observed in fractions
examined.
Cell cycle related variation in the surface morphology of Chinese hamster
ovarian (CHO) cells in culture has been reported by Porter et al. (1973). Their
obsei"vatious differ from those in this report and this may be due to the different
146
L. CHING, J. GAVIN, ]. MARBROOK AND M. SKINNER
modes of growth. Presumably our mastocytoma cells, growing free as an ascitic
tumour, were not influenced by the constraints imposed on GHO cells which
grew as monolayers attached to a surface.
Recent reports have proposed that T lymphocytes can be distinf^uished from
B lymphocytes on the basis of their tt)pography (Polliack et ai, 1973) but this
conclusion has not been universally accepted (Thurman, Buur and Goldstein,
1975). Our observations suggest that cell surface morphology may be an
unreliable criterion unless maintained throughout the cell cycle.
Despite the marked differences in the appearance of the cell surface, the
fractionated cells were indistinguishable from unfractionated cells in their susceptibility to cell-mediated immune lysis. It thus appears that the sensitivity of
the mastocytoma cell-mediated lysis does not vary significantly during the cell
cycle. This is interesting, as fluctuations in the expression of surface H2 antigens
during the cell cycle have been reported in several cell lines. Cikes and Friberg
(1971) noted that antigen concentration was highest during G] and lowest
during S-phase. The sensitivity of cells to antibody-mediated lysis does correspond to cyelic fluctuations in antigen concentration in cultured mastocytoma
cells (Pasternak et ai, 1971) and mouse lymphoma cells (Gikes and Friberg.
1971). These cells were most sensitive to anti-H2 sera in G, and then decreased
in sensitivity during the S-phase as the antigen concentration decreased. In
contrast, a human Iymphoid cell line was found not to vary in sensitivity to
cytotoxic ajitibodies despite cyclical variations in aiitigen concentration (Pellegrino et ai, 1974), whereas YGAB murine tumour cells varied in sensitivit)'
to antibody-mediated lysis when no difference in antigenic expression could be
detected (Lerner et ai, 1971).
There are probably many factors other than antigenic expression which
influence the sensitivity of the eells to immune lysis. The present investigation
suggests that there is no great variability in the sensitivity of mastocytoma cells
to cell-mediated cytolysis during the cell cycle. It may be that the area of
contact between the killer and target cell is such that only substantial differences
in antigen concentration wonld contribute to differences in the efficiency of
cell-mediated lysis.
Acknowledgements. This work v\'a.s supported by the Medical Research Council of New
Zealand, and in part by the Auckland Division, Caneer Society of New Zealand Inc.
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