(CANCER RESEARCH 36, 3131-3137, September 1976]
Cell Cycle and Morphological Changes during Growth and
Differentiation of a Rat Basophilic Leukemia Cell Line
Donald N. Buell,1 B. J. Fowlkes, Henry Metzger,
and Chaviva lsersky2
Immunology Branch fD. N. B.) and Laboratory of Pathology (B. J. F.J, National Cancer Institute, and Section on Chemical Immunology, Arthritis and
Rheumatism Branch, National Institute of Arthritis, Metabolism and Digestive Diseases (H. M. , C. I.J, NIH, Bethesda, Maryland 20014
SUMMARY
Cells of the rat basophilic leukemia cell line RBL-1 differ
entiate maximally when permitted to achieve growth arrest
in a high-density stationary phase, in which the cell number
is constant, and the cells are arrested in a G phase of the
cycle. Features of differentiation are the accumulation of
large basophilic granules and increases in membrane me
ceptons for immunoglobulin E. However, changes in hista
mine content did not parallel granule development on
changes in immunoglobulin receptor concentration. During
rapid “forced
exponential―growth, the cell number doubles
every 8 hr, 50% of the cells are in S phase, and diffenentia
tion is minimal.
stainedas above.
INTRODUCTION
A transplantable basophilic leukemia, induced in a Wistar
mat by administration of /3-chlonoethylamine p.o. (7), has
been adapted to tissue culture (19). Leukem mcbasophils
from both the tumor and the tissue culture-adapted line
(RBL-1) have the characteristic features of differentiated
basophils: prominent basophilic granules, substantial his
tamine levels, and large numbers of receptors for IgE (19).
These unique cells have by now been used extensively by
ourselves and others. It seems appropriate, therefore, to
describe more completely the growth patterns of these
cells. We have found that the cells undergo striking vania
tions in their degree of differentiation, as monitored by
basophilic granule development, and in their numbers of
receptors for IgE. This paper describes the relationship of
morphological differentiation to variations in cell cycle pa
nametens. Changes in the receptors for IgE during the cell
cycle have been described in detail elsewhere (17).
MATERIALS AND METHODS
Cell Culture. Cultures were grown as described previ
ously (17) using 20% fetal calf serum. Tylocine, an anti
pleuropneumonia-like organism agent (Grand Island Bio
logical Co., Grand Island, N. Y.), was added to culture
media during the course of these experiments when Myco
, Presentaddress:Divisionof HematologyandOncology,Department
of
Medicine, Childrens Hospital Medical Center, Boston, Mass. 02115.
2 To
whom
correspondence
plasma was cultured from other cell lines in the laboratory.
No effect of Tylocine was noted on the growth of RBL-1
cells. Cells were grown in 30- on 250-mI Falcon flasks on in
spinner flasks.
Conventional Cytology. Cells were counted in a standard
hemacytometer and viability determined by 0.08% trypan
blue exclusion. Freshly prepared unfixed smears were pre
pared using a cytocentnifuge (Sakuna Fine Technical Co.,
Tokyo, Japan) at 500 rpm and were stained with Wright
Giemsa stain on an Ames Hematek slide stainer (Ames Co.,
Division of Miles Laboratory, Inc., Elkhart, Ind.) by the Clini
cal Hematology Service, Clinical Center, NIH. Chromosome
preparations were made from exponential cultures after a
30-mm exposure to Colcemid, 0.04 @g/ml(Grand Island
Biological Co.), using the method of Puck et a!. (25) and
should
be
addressed.
Received July 30, 1975; accepted May 25, 1976.
Scanning Electron. Micrography. These studies were
kindly performed by Dr. Raul Braylan, National Cancer Insti
tute, Bethesda, Md. Washed cells (2 x 106/ml) were fixed in
cold 2.5% glutanaldehyde, pH 7.4, for at least 2 hr. Cells (1 x
10w)were collected onto Flotnonic filter membranes (Selas
Flotnonic, Spring House, Pa.) of 0.45-Mm pore size. The
filters were rinsed, dehydrated in graded alcohols and una
nyl acetate, and then, by the critical point method, coated
with gold-palladium on a tilted rotatory stage, examined
with an ETEC Autoscan at 20 kV and 45°tilt, and photo
graphed with Polaroid 52 film.
Cell Cycle Determination. [3H]Thymidine, 0.36 Ci/mmole
(Schwarz/Mann, Orangebung, N. V.), was added to cultures
to a concentration
of 1 mCi/mI.
Pulse-labeled
samples were
processed after 15 mm for determination of the S phase by
the “labeling
index―(22). Continuous [3H]thymidine label
ing was used to determine the length of the G phase ac
cording to the technique of Maekawa and Tsuchiya (22).
The percentage of labeled mitoses was used to determine
the length of the G2 phase (25). Slides were prepared for
autoradiography by the technique used for chromosome
analysis (25). They were dipped in NTB-2 emulsion (East
man Kodak Co., Rochester, N. Y.), dried, and stored at 4°.
After 6 days of exposure they were developed in Kodak D-19
developer, fixed, and stained @nthe Ames slide stainer.
Nuclei with more than 10 overlying grains (x3 background)
were scored as labeled.
Automated Cytology. Cell counts and volume distribution
curves were obtained using a Model B Coulter counter
equipped with a Coulten channelizer and an X, Y plotter
(Coulter Electronics, Hialeah, Fla.). Analysis of relative sin
SEPTEMBER1976
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3131
D. N. Buelleta!.
gle-cell DNA content was performed by FMF2 using the
mithnamycin method on a Los Alamos Scientific Laboratory
multiparameter cell sorter (5, 6, 31). For the latter studies
cells were centrifuged at 1000 rpm, washed with filtered
0.85% NaCI solution , fixed in 75% ethanol containing 15 mM
MgCl2, and stored at 4°until analyzed. For the FMF analysis,
2 x 10@cells were suspended in 4 ml of 0.15 M NaCl-0.01 M
NaPO4,pH 7.4, containing mithnamycin, 100 @g/ml.After 20
mm
at room temperature, samples were nun on the cell
sorter, and the oscilloscope pattern of single-cell DNA
quantitations was recorded on Polaroid type 105 positive
negative film (Polaroid Corp. , Cambridge, Mass.).
The FMF patterns were converted to digital form by pro
jecting the polaroid transparencies on graph paper using an
enlarger at a fixed setting. Relative numbers of cells con
taming G1,5, and G2quantities of DNA were estimated from
the distributions using the Simulation, Modeling and Analy
sis 26 computer program (2, 3) with constraints similar to
those used by Dean and Jett (6). However, the inter-G1-G2
(S-phase) distribution curve was not constrained to fit a
2nd-order polynomial but instead was assumed to result
from a series of 10 gaussian distributions with means and
spreads evenly spaced between the extremes determined
for the G, and G2 populations.
Thymidine Block and Synchronization. To achieve
growth arrest in S phase (13), thymidine was added to a
culture to give a final concentration of 2.5 mM. Synchroni
zation by double thymidine block was accomplished by a
modification of the technique of Galavazi et a!. (13). Cells in
culture were exposed to 2.5 mM thymidine for 8 hr, centni
fuged for 10 mm at 130 x g at room temperature, washed in
medium with 10@ M deoxycytidine, (Schwarz/Mann), me
centrifuged, and suspended in medium without nucleo
tides. After 12 hr 2.5 mM thymidine was again added for 8
hn, and the cells were centrifuged, washed in fresh medium,
centrifuged, and resuspended in medium with 10_6M deox
ycytidine.
Separation of Cells by Velocity Sedimentation on Ficoll
Gradients. The technique described for human lymphoid
cells (9) was modified for RBL-1 cells. Gradients (5 to 10%,
w/v) were prepared by dissolving Ficoll (Pharmacia Fine
Chemicals, Piscataway, N. J.) directly in bicarbonate-free
Eagle's Spinner No. 1 medium containing 0.01 M N-2-hy
d roxyethylpiperazine-N ‘-2-éthanesulfonic
acid buffer, peni
cillin, streptomycin, Tylocine, and glutamine but no serum.
The solutions were sterilized by filtration through a Nalgene
filter unit with a 0.45-.@m membrane (Nalge Sybron Co.,
Rochester, N. V.). A 5 to 10% linear continuous gradient
was prepared using a Buchlen Instrument density gradient
generator. The final gradient was 80 ml in a 3- x 10.5-cm
cylindrical No . 32086 polycarbonate tube (International
Equipment Co. , Needham Heights, Mass.). An additional 5
ml of a 5% Ficoll solution were layered on top of the gra
dient. A monodisperse cell suspension in medium contain
ing 2 to 3 x 10@cells at 5 x 106 cells/mI was layered on top
of the gradient. The samples were centrifuged for 30 mm at
80 x g at room temperature in an International PR-2 centni
fuge using a swing-out rotor. Fractions of 4 ml each were
tube down through the gradient. A polyethylene tube was
attached to the stainless steel tube, and the fractions were
collected by siphoning. The cells in the gradient fractions
were counted, their volume distribution was determined,
and the distribution of DNA content was assessed by FMF
analysis.
RESULTS
General Characteristics of Cell Line. In stationary flasks
RBL-1 cells generally form a sparse monolayer with overly
ing cells in suspension proliferating to high densities. No
such attachment occurs in spinner flasks with the cells kept
in continuous motion, and the cells reach densities of 2 x
106 cells/mI or higher. Reproducible growth patterns, high
viabilities in prolonged stationary growth, and high levels of
receptors for IgE were found using medium supplemented
with 20% fetal calf serum.
During routine maintenance feeding schedules, RBL-1
cultures undergo a pattern of a lag (12 to 36 hr), 1 to 3
doublings, and then a stationary phase where they can be
maintained for 48 to 96 hr with high viability (>90%). When
stationary-phase cells are centrifuged and resuspended in
fresh medium at 3 x 10@cells/mI, they undergo a lag phase
during which the cells increase in volume and pass through
the G1, 5, and G2 phases of the cell cycle in partial syn
chrony as indicated by FMF analysis of their DNA content
(Chart 1). After the exponential phase the cells enter a
prolonged stationary phase with a decreasing average cell
volume. Such cells have the G content of DNA (Chart 1).
RBL-1 cells could be kept in “forced
exponential―growth
by removing two-thirds to three-fourths of the cell suspen
sion daily and restoring the volume in the spinner flask with
fresh medium. When cells that had been in rapid exponen
tial growth for a few generations were suspended in fresh
medium at 3 x 10@cells/mI without further feeding, the
characteristic pattern of growth into a high-density station
ary phase occurred. However, after 8 to 10 days of forced
exponential growth, such cells lose the capacity to achieve
a high-viability prolonged stationary phase. The cells grow
quickly to high densities and die, apparently before un
dergoing the metabolic alterations necessary for prolonged
stationary phase survival.
Lag-phase cells enter S phase between 9 and 16 hr after
resuspension in fresh medium. In other cultures the length
of time prior to the start of S phase varied, being longer for
cultures with longer stationary phases. Similarly, the total
duration of the lag phase (range, 16 to 36 hr) appeared to
correlate with the length of time during which the cells were
in stationary phase. We have not determined whether the
length of time in S phase is also variable and contributes to
the variation in the duration of the lag phase on whether the
main variability is in the time that elapses before the ‘
‘
rest
ing―
cellscan reenterthe growth cycle.
Cell Cycle Parameters. Using [3H]thymidmneincubation
and autonadiognaphy, the durations of the cell cycle and its
G1, 5, and G2phases were determined for a rapidly growing
“forced
exponential―culture with a doubling time of 8 hr. A
collectedby carefullyplacinga 0.2-x 15-cm stainlesssteel 15-mm pulse of [3H]thymidine labeled 50% of cells (labeling
index, 0.5). Since the cell cycle time, T(., = 8 hr, the duration
2 The
abbreviation
used
is: FMF,
flow
microfluorometry.
3132
CANCER
RESEARCH
VOL. 36
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Basophi!ic Leukemia Cell Changes
1) indicated that the cells were in S phase 2 and 4 hr after
release from thymidine block and then passed through G.,
before mitosis. In a 2nd double thymidine-synchronized
Ohr
culture, FMF analyses of single-cell DNA content distnibu
tions were included. At 10 hn, just after the 1st synchronous
division and cell doubling, all cells had the G content of
DNA. Subsequently, the cells passed parasynchnonously
through S and G2 as indicated by the DNA patterns.
Gradient Fractionation by Rate Zonal Centrifugation.
ci:
Three different cultures, a forced exponential culture and
w
cultures analyzed at the late exponential and early station
::i
ary stages, were analyzed. The cell distribution profiles
z
differed for cells from different stages of growth with the
-J
-J
@jr
larger cells of the “forced
exponential' ‘
culture penetrating
w
0
deepen
into
the
gradient
than
those
of
a transitional or
w
>
stationary culture. Chart 2 presents analyses of cultures
I—
sampled in late exponential and early stationary stages of
-J
growth. Chart 2a shows the Coulter counter volume distni
LiJ
ci:
butions for the 2 cultures. Each of these shows a considena
ble spread of cell volumes within the parent cultures. Chant
2b showsthat the individual gradient fractions show a much
narrower distribution of cell volumes and that the depth of
penetration into the gradient correlates directly with the cell
volume. Chant 2, c and d, presents the DNA distribution
pattern of the parent cultures and of the derived fractions.
Analysis of the distribution of cells in the different cell cycle
phases derived from the FMF data showed that separation
TI@L:
by size achieves separation by cell cycle parameters, with
CHANNEL NUMBER
the larger G2-phase cells enriched in the bottom fractions,
(Relative DNA content)
G cells predominating at the top, and S-phase cells en
Chart 1. FMF analysis of DNA content in cells from a culture during a lag
niched in the intermediate fractions (Table 2). Although the
phase of growth. At 0 time the cells had just been diluted to 3 X 10' cells/
ml after 3 days of stationary growth at 2 x 10@cells/mi. No change in cell
analysis of the parent culture in the early stationary phase
number was observed till 31 hr, at which time the cells numbered —4x 10'
showed no significant numbers of G2 cells, the gradient
cells/mI.
fractionation demonstrated their presence. The G cell con
tamination in the lower fractions probably results from
of S phase,
—4 hr (from labeling index = Js/J(). Labeled “trailing―
due to the large excess of G cells in the parent
mitoses were absent after 1 hr of exposure to [3Hjthymidmne, culture, G cells that sedimented as doublets, M cells com
but 95% of mitoses were labeled by 2 hr; thus, the duration
pleting mitosis during on after centnifugation (at room tem
of G,
= —1
.5 hr. The length of G was determined
perature), large G cells ready to enter S phase, or cells
directly by following the disappearance of unlabeled cells carried down by passing the sampling tube through the
from the culture during continuous
exposure to gradient.
[3H]thymidine (22). After 1 to 2 hr no unlabeled cells entered
Morphological Differentiation. RBL-1 cells vary in ap
mitosis. The decrease in unlabeled cells after that time pearance from primitive cells resembling pnomyelocytes to
serves as a measure of the passage of cells from G into S
phase. Between 1 and 4 hn, the percentage of unlabeled
Table 1
cells fell to 0. Thus G was 2 to 3 hr long. The rate of entry of
cells into S (—50%in 4 hr) was consistent with an 8-hr cell Cell count and DNA synthesis after release from double thymidine
block
cycle time. The viability of the forced exponential cultures
in
was 100@/@
and all cells were cycling rapidly, as indicated by
(x
in
corporation―
100% labeling of cells with [3H]thymidmne in 4 hr.
Time10@)07.727.70.8212.347.80.8116.877.90.355.21410.54.92415.80.255
(hr)Cells/mI 10')Labeling
dex[3H]Thymidine
(cpm x
Cell Cycle Synchronization Studies. Cultures that were
exposed to excess thymidine for prolonged periods of time
showed a state of ‘
‘
unbalanced growth' ‘
in which the cells
grow but do not synthesize DNA or proliferate (21, 29). FMF
analysis confirmed that the cells were still in early S phase
GI
(a)
@
@
G2
H@1
even after25 hr.Cellsexposed to excess thymidine twice
for 8 hr with an interval of 12 hr were similarly arrested but
resumed growth upon elimination of the thymidine and
addition of deoxycytidmne. [3H}Thymidine incorporation me
suIts and [3H]thymidine pulse autoradiography data (Table
SEPTEMBER
‘1Counts
incorporated
by
1
x
10@
cells/mI
incubated
for
15
mm
with 1 mCi of [3H]thymidmne per ml, spun down, washed with 0.15 M
NaCI-0.01MNaPO4(4°),
dissolvedin 1 ml Hyamine,and counted.
1@Cultures
approaching
stationary-phase
conditions.
1976
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1976 American Association for Cancer Research.
3133
D. N. Buell et al.
d
G1 G2
a
:3f.A
l2J@,
copy
by Dr. K. Becker.
The
photographs
were
in addition
kindly reviewed with us by Dr. R. Hastie. The cells show
many of the features seen in normal human basophils (15)
with no unusual features; no viral-like bodies were ob
served. The cells have also been examined by scanning
electron microscopy (Fig. 2c). The predominant topology
consists of hundreds of villi, fine veil-like surface protnu
sions being a less common feature. Cells from a “forced
_______
exponential―
and
astationary-phase
culture
appeared
simi
z
:3
z
1211109876
lamby scanning electron microscopy.
Chromosome counts were performed on 20 cells. Five
gave a tetraploid value of 84 while the remainder were
hypotetnaploid, 1 cell having as few as 70 recognizable
ill
13121110
87
_______chromosomes.
Despite
theheteroploidy,
single-cell
DNA
:ok-
b
8@
i7@
6
2
8
24
6
2
8
24
CHSNN[L
NUMBER
RELATIVE
DNACONTENT
Chart2. Left: volume distribution spectra of cells from late exponential
(ExpO) and early stationary
(Stat) cultures
(a) and in Ficoll gradient
fractions
of these 2 cultures (b). Numbers over peaks, fraction numbers. All ordinate
scales are linear. Right: c, distribution of cells in G, 5, and G, + M phases of
the cycle in a late exponential culture and in Ficoll Gradient Fractions 6 to 12
from it; d, the distribution in an early stationary culture and in Ficoll Gradient
Fractions 7 to 13 from it. The ordinate and abscissa scales are linear.
well-differentiated basophils, depending on growth condi
tions (Figs. 1 and 2). The content and size of basophilic
granules in RBL-1 cells varied depending on the state of
growth of the culture. Cells from “forced
exponential―cul
tures uniformly had only a few small granules (Fig. la). The
appearance on a Wnight-Giemsa-stained smear was that of
primitive cells of myeloid origin (promyelocyte) with small,
sparse granules, so that the cells could not be easily identi
fied as basophils. Approximately 50% of the cells in such
cultures are passing through the G phase of the cell cycle.
As the cultures entered and remained in stationary phase,
basophilic granules increased in number and became more
prominent. In late stationary phase, these cells uniformly
had the appearance of well-differentiated basophils with
large granules (Fig. lb). As has been shown, these cells
have a G1 DNA content.
When stationary-phase cells were resuspended in fresh
medium at lower density (lag phase), the basophilic gran
ules remained intact until the 1st cell division (Fig. 2a).
Thereafter, granules decreased in size and number as the
cells passed through successive divisions (Fig. 2b). During
routine maintenance, with twice-weekly feeding of the cul
tune, the cells remained fairly well differentiated, with prom
inentgranules.
Granule development was dependent upon growth of the
culture into high-density stationary phase. When growth of
an exponential culture was arrested with excess thymidine,
granule development was that characteristic of the starting
exponential culture, even after prolonged thymidine arrest.
Shortly after we received the tumor, some thin sections of
cells from tumor minces were examined by electron micros
3134
quantitation using FMF analysis revealed characteristic pat
temns representing the G, 5, and G2 populations so that
these cells appear to have a stable DNA content (18). No
further kanyotyping has been performed.
Histamine Content. A limited number of cultures were
followed for histamine content during culture growth using
an automated procedure (28). The results of 1 such expeni
ment are given in Table 3. It can be seen that in exponential
growth there was no decrease in histamine content per ce!!
despite the rapid divisions; if anything there was a small
increase. Several other cultures showed qualitatively similar
results except that fairly substantial decreases in histamine
content were usually seen in the latter part of the exponen
tial phase.
We never observed a substantial increase in histamine in
the stationary phase of culture growth comparable to the
Table 2
Analysisof cells from early
aFicoll
stationary culture fractionated on
gradientFMF
(%)Fraction
MTotal
analysis
Cells/mi
(x 10 ‘)
G1
S
69
29
0.84
1.30
1.91
2.90
18
29
72
92
48
65
28
8
1.90
99
28
349
610
011
012
013
G2 +
1
0.87 0Table 100
0
3Histamine
content of cultured RBL-1
cellsHistamineCells/mI
(ng/106Culture
cells)Early
stage
50024-hrstationary
stationary
46048-hr
47096-hr
stationary
300Lag
stationary
430Lag
530Start
600Midexponential
exponential
670Forced
Time (hr)
72
96
120
144(0)―
148(4)
156(12)
162(24)
192(48)
500Forcedexponential
exponential
120
690Forced
730‘I exponential
After
dilution
with
fresh
144
216
(x l0@)
2.2
2.1
2.1
2.2 (0.3)―
0.3
0.3
0.33
1.0
0.95
1.0
0.8
medium.
CANCER
RESEARCH
VOL. 36
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Basophi!ic Leukemia Cell Changes
observed morphological changes on increases in receptors
for IgE (see below). The histamine values shown in Table 2
are similar to those most frequently observed oven the
course of the 1st 1.5 years that the cells were cultured.
Recently, our cultured cells have shown substantially lower
histamine contents.
DISCUSSION
Leukemic basophils of the RBL-1 cell line can be induced
to undergo reproducible
patterns of growth and diffenentia
tion . The length of the G , 5, and G, phases of the cell cycle
was determined only for the “forced
exponential―culture.
Under these conditions, all the cells cycle rapidly and the
phases have minimal variation (26). Under any other condi
can be considered to be arrested in the G phase on to be
resting “G0―
cells (8, 10, 20). However, G cells in these
instances may have markedly different properties than do
the G-phase cells of a cycling, exponentially growing cul
tune (1). This is clear from our results where G cells from an
exponential culture have markedly decreased basophilic
granules and IgE receptors (17) compared with G cells of
similar size from stationary-phase cultures.
The nature of the transition from stationary phase to
reentry into the cell cycle should be examined further. Dun
ing the usual 20- to 24-hr lag in growth, up to 16 hr may
elapse before the cells enter the S phase. This may be the
time necessary for the cells to revert from a G, or “station
any-phase G―and to reenter the G phase of an active cycle
(1, 8, 10). A block in progression to terminal differentiation,
the state where the cells are unable to reenter the cycle,
tions, determination
of the lengths of G, S and G2 phases
appears to be a feature of leukemias (14) but is not absolute.
has little meaning with changes in cycle parameters taking
Friend virus-induced munine emythnoleukemias (12) and
place more rapidly than they can be studied by such tech
granulocytic leukemias of mouse (11) and man (23) can be
niques as percentage-labeled mitosis curves (26, 27).
induced to undergo terminal differentiation in vitro. Addi
The growth patterns have been correlated with variations
tional data would be required to determine whether RBL-1
in cell cycle parameters. Stationary-phase
cells are small,
cells in prolonged stationary-phase culture even reach the
are arrested in the G phase of the cell cycle, and undergo
state of terminal differentiation.
differentiation as manifested by large prominent basophilic
The differentiation of normal basophils has been studied
granules and 3- to 5-fold increases in the IgE receptor in guinea pig bone marrow during a serum-induced baso
concentration
(17). By contrast,
cultures kept in rapid
‘
‘forcedexponential― growth have minimal differentiation
with fewer, smaller granules and lower levels of IgE necep
tons. Paradoxically, the histamine content, which might be
considered a differentiated characteristic of the cells, is
quite low in the stationary-phase cultures. We are uncertain
as to the cause of this apparent discrepancy. Stationary
cells are more fragile. It is more difficult to make good cell
smears from such cultures, and they show an increased
leakage of histamine when incubated in test tubes. This may
partially explain the lower values observed for the stationary
cells. Attention should also be called to the fact that the
histamine content has varied greatly. Although many of the
cultures have values such as 400 to 600 ng/106 cells, occa
sionally values as high as 1050 to 1400 ng/106 cells were
seen; however, values as low as 50 to 150 have been seen
more recently. We are unable to explain satisfactorily these
oscillations.
In 5- to 6-day-old Sarcoma 180 cultures, characterized by
high density and decreasing
growth
rates, traditional
tech
niques of autoradiography following pulse [3H]thymidine
labeling (27) give varying results, depending on such fac
tons as duration of emulsion exposure and grain count
threshold. In those studies, correlations of cycle parameters
determined
using [3H]thymidine
phil response (30). Studies in other species have been less
extensive because of the relative paucity of basophils in
normal marrow (32). These electron microscopic studies
were based on identification of cells as basophils by granule
appearance and correlations with the degree of nuclear
maturation.
Rat mast cell differentiation
has also been stud
ied (4, 24), but again the order of events had to be inferred
and could not be directly followed. Precise time sequence
studies of basophil differentiation are impossible using
such techniques. To the extent that the RBL-1 leukemic
basophil cultures mirror normal basophil (?mast cell) differ
entiation, detailed studies should now be possible.
ACKNOWLEDGMENTS
We are grateful to Dr. F. K. Millar (Laboratory of Theoretical Biology,
National Cancer Institute) for developing the computer program used to
analyze the FMF data, to Dr. R. Siraganian (Laboratory of Microbiology and
Immunology. National Institute of Dental Research) for performing the hista
mine analyses, to Dr. Raul Braylan for the scanning electron micrography,
and to M. Cassidy and Dr. C. Herman, Cytology Automation Program (Labo
ratory of Pathology, National Cancer Institute) for their cooperation and
assistance.
REFERENCES
incorporation-autoradiog
raphy with cell cycle determinations based on single-cell
DNA quantitation by FMF analysis revealed low levels of
DNA synthesis in cells with the G, content of DNA (27). In
such cultures, S phase does not appear to exist as tradition
ally defined (16); low levels of DNA synthesis may occur
throughout most of the cell cycle. A similar situation may
exist in RBL-1 cultures in stationary phase, where all the
cells appeared to be in G by FMF analysis but low levels of
[3H]thymidine incorporation were still measurable (17). In
these RBL-1 stationary-phase cultures, as mother cultures
that manifest high-density growth inhibition (16), the cells
1. Backer, H., Stanners, C. P., and Kudlow, J. E. Control of Macromolecu
lar Synthesis in Proliferating and Resting Syrian Hamster Cells in Mono
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of the Boundary between
CANCER
RESEARCH
VOL. 36
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Basophi!ic Leukemia Ce!! Changes
‘41
la
Fig. 1. a, cellsfrom a “forced
exponential―
culture:b, cellsfrom a late-stationary-phase
culture.Wright-Giemsa-stained
cytocentrifugepreparations.Final
magnification. x 630.
2a
2b
Fig. 2. a, cells after 21 hr in lag phaseof growth: b. cells from sameculture as in a after -13 hr of exponentialgrowth (37hr after resuspension)a, b,
Wright-Giemsa-stained cytocentrifuge preparations photographed at x 320 and magnified x 2.5 from negatives); c, scanning electron micrograph of cells
from a stationary culture. Final magnification, x 5300.
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3137
Cell Cycle and Morphological Changes during Growth and
Differentiation of a Rat Basophilic Leukemia Cell Line
Donald N. Buell, B. J. Fowlkes, Henry Metzger, et al.
Cancer Res 1976;36:3131-3137.
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