[CANCER RESEARCH 42, 445-449,
0008-5472/82/0042-OOOOS02.00
February 1982]
Induction of Differentiation of HL-60 Cells by Dimethyl Sulfoxide:
Evidence for a Stochastic Model Not Linked to the Cell Division
Cycle1
Corrado Tarella,2 DarÃ-oFerrerò, Eugenio Gallo, Giovanni Luca Pagliardi, and Francis W. Ruscetti3
Laboratory of Tumor Cell Biology, National Cancer Institute, NIH, Bethesda, Maryland
Patologia Sp. Medica, Università di Torino. Torino, Italy [D. F., E. G., G. L. P.]
ABSTRACT
Induction of differentiation of the human promyelocytic leu
kemia cell line HL-60 by dimethyl sulfoxide (Me?SO) was ana
lyzed to determine the relationship between exposure time of
the inducer and cell cycle. A minimum incubation time of 12 hr
with Me;.SO was required in order to induce differentiation in a
small but significant proportion of cells. These expressed dif
ferentiation markers (morphology, phagocytosis, and nitroblue
tetrazolium reduction) up to 12 hr after Me2SO was removed
from the medium. For periods beyond 12 hr and as long as
120 hr of contact of HL-60 cells with the inducing agent, a
linear rise in the percentage of differentiated cells was ob
served. The sensitivity to Me2SO of HL-60 cells synchronized
by double thymidine block was examined and was found to be
similar to that of nonsynchronized cells. The effect of Me2SO
was not altered when incubated with cells at different phases
of the cell cycle. Finally, even nonproliferating cells were sen
sitive to the inducing effect of Me:>SO. The data are consistent
with a stochastic model of induction to differentiation without
having any linkage to the cell cycle.
INTRODUCTION
The HL-60 cell line, derived from a human promyelocytic
leukemia (4), is a unique human leukemic cell line capable of
terminal differentiation in the presence of several inducing
agents (3, 5). Me2SO4 is one of the most studied differentiation
inducers. The morphological, biochemical, and immunological
changes that it induces in HL-60 cells have been described
recently (2, 7, 11). The leukemic promyelocytes of HL-60,
when cultured in the presence of Me2SO, differentiate into
more mature cells of the myeloid lineage, displaying functional
characteristics associated with normal peripheral blood granulocytes such as chemotaxis, phagocytosis, and Superoxide
anión production (6, 18).
In this study, we have studied: (a) how long an exposure to
Me2SO is necessary to induce differentiation of HL-60 cells;
Ob)whether the differentiation of induced cells is an irreversible
process that continues in the absence of the inducer; and (c)
' Supported in part by Regione Piemonte and S. Giovanni Hospital. Torino,
Italy, and by CNR Grant 780283596, Progetto Finalizzato per la Crescita Neo
plastica.
2 Present address: Cattedra di Ematologia, Università di Torino, Torino. Italy.
3 To whom requests for reprints should be addressed.
4 The abbreviations used are: Me?SO, dimethyl sulfoxide; MEL, murine erythroleukemia; RPMI 1640. Roswell Park Memorial Institute Medium 1640: NBT.
nitroblue tetrazolium; Ml, mitotic index.
Received July 8, 1981 : accepted October 27, 1981.
FEBRUARY
1982
20205 1C. T., F. W. R.], and Cattedra di Ematologia
and Cattedra di
whether the commitment to differentiation by Me2SO is related
to the cell cycle.
Previous studies of this type using Friend MEL cells have led
to contradictory conclusions regarding the requirement of DMA
synthesis for differentiation of MEL cells (8, 10, 12, 13, 15,
17). We have found that proliferation is not required for myeloid
differentiation. In addition, the data reported here are consist
ent with a stochastic model similar to the one reported in Friend
cells (9, 19, 22) governing the transition from the uncommitted
to the committed state. For this transition, a particular phase of
the cell cycle does not seem to be required, nor is,there a need
of cellular proliferation.
MATERIALS AND METHODS
Cell Lines and Culture Conditions. HL-60 cells were passaged
twice weekly in RPM11640 (Grand Island Biological Co., Grand Island,
N. Y.) supplemented with 10% heat-inactivated fetal calf serum (Rerteis,
Phoenix, Ariz.) in 7% CO2 atmosphere at 37°. The experiments were
performed on cells between passages 30 and 48.
Induction of differentiation was obtained by seeding the cells at a
concentration of 5 x 105/ml in the presence of Me2SO (Microbiological
Associates, Rockville, Md.) at a final concentration of 1.2% (v/v) for
various periods of time. After exposure, the cells were resuspended in
Me:.SO-free medium.
Cell counts were performed in a Burker chamber, and viability was
assessed by trypan blue dye exclusion. For morphological assessment
of the cells, Cytospin slide preparations of aliquots of cell suspensions
were prepared using a Cytospin centrifuge and stained with WrightGiemsa.
NBT Reduction Test. The ability of cells to generate Superoxide
anión was demonstrated by the reduction of the soluble NBT to blueblack insoluble formazan (25). One ml of cell suspension was incubated
for 20 min at 37°with an equal volume of 0.2% NBT (Sigma Chemical
Co., St. Louis, Mo.) dissolved in phosphate-buffered
saline (pH 7.2;
0.15 M without Ca**, Mg+*), in the presence of 200 ng of 12-Otetradecanoylphorbol-13-acetate,
obtained from the NIH carcinogen
repository (6). Scores of NBT-positive cells were performed on at least
500 cells.
Phagocytosis of Activated Yeast. Yeast was activated by incubation
with fresh human serum at a concentration of 2 x 108 particles/ml, for
20 min at 37". Yeast particles were then resuspended in phosphatebuffered saline at a concentration of 10" ml. Cells were incubated in
RPM11640 at a concentration of 106/ml, with activated yeast particles
(I07/ml) for 30 min at 37°. The percentage of cells which have
phagocytosed was determined in a hemocytometer by counting those
containing one or more yeast particles in their cytoplasm on at least
500 cells (14).
Synchronization of Cells. HL-60 cells were synchronized with re
spect to the cell cycle by a modification of the method described by
Puck (21 ). Briefly, cells were incubated for 24 hr in medium containing
2 niM thymidine and then washed and resuspended in thymidine-free
445
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C. Tarella et al.
medium for 10 hr. They were then again blocked by incubation with
thymidine for 16 hr. Release from the block was obtained by resuspension in fresh medium without thymidine.
The rate of synchrony achieved was monitored by removing aliquots
of cultures at 60-min intervals for determination of DNA synthesis, Ml,
and cell number. DNA synthesis was determined by incorporation of
[3H]thymidine. Cells were incubated at a concentration of 2 x 105/ml
with [3H]thymidine (3 /iCi/ml; specific activity, 5 Ci/ml) for 30 min at
37°.They were then washed and precipitated with 10% trichloroacetic
acid; the precipitate was collected on Millipore filters, washed with 5%
trichloroacetic
acid, and counted in Instagel (Packard) in a liquid
scintillation counter. The Ml was determined by counting the mitotic
figures in 1000 cells on a smear prepared with Cytospin centrifuge and
stained with Wright-Giemsa. The length of cell cycle phases was
determined from DNA synthesis, Ml, and cell density, as described
Volpe and Volpe (29).
by
RESULTS
Cell Growth and Me2SO-induced Differentiation in Nonsynchronized HL-60 Cells. The length of time of contact with
Me2SO necessary to induce differentiation was determined by
culturing HL-60 cells in the presence of Me2SO for 6, 12, 18,
24, 30, 44, 72, 96, and 120 hr.
All cultures were initiated at a cell density of 5 x I0b cells/
ml. After 2 days, a density of 1.1 to 1.3 x 106 cells/ml was
achieved in all cultures. By the third day, the growth rate varied
depending on the length of exposure to Me.,SCX Cells main
tained with Me2SO for 6 hr achieved a density of 2.5 x 106/ml
at Day 5, while there was a progressive decrease in growth at
longer exposure times to Me2SO (Chart 1). Viability was over
90% at days 1 and 2 and between 80 and 90% from Day 3 to
Day 5.
The pattern of induction of HL-60 cells by Me2SO was
followed for 6 days, with Day 0 as the first day of exposure. As
shown in Chart 2, exposure for at least 12 hr was needed to
produce minimal increase in the proportion of NBT-positive
cells, while an increasing proportion of mature cells was then
observed up to the 120th hr.
Cells exposed to Me2SO for 12, 18, 24, and 30 hr showed
a maximum peak of percentage of NBT-positive cells (14, 22,
35, and 58%, respectively) after 2 days of culture; cells ex
posed for 44 hr achieved their peak after 3 days (86%); the
highest proportion of NBT-positive cells was observed on Day
4 in cultures exposed for 72 hr (92%) and on Day 5 in cultures
exposed for 96 and 120 hr (96 and 99%, respectively). A
decrease in the percentage of differentiated cells was observed
in all cultures after the maximum differentiation peak. Presum
ably, this is due to proliferation of nondifferentiating cells.
A small percentage of noncommitted cells (1 to 6%) was
even present in cultures exposed to Me2SO for as long as 120
hr as shown in Chart 2. These cells when transformed to the
Me2SO-free medium were able to reconstitute the normal pop
ulation of self-renewing HL-60 and displayed a normal Me2SO
sensitivity pattern.
The differentiation pattern observed using the ability to phagocytize activated yeasts as a functional criterion showed a
curve similar to that observed with the NBT reduction test. The
maximum percentage of cells capable of phagocytizing was
reached 15 to 20 hr earlier (Table 1).
Morphologically differentiated cells appeared only after 2
days of culture, and their percentage increased, prolonging the
time of exposure to the inducer. A comparison of phagocytosis
and NBT reduction activities with the morphology of HL-60
cells treated for 30 hr with Me2SO is shown in Table 1.
Synchronization of Cells. Synchronization of cells with re
spect to the cell cycle was obtained by a double thymidine
block as described in the "Materials and Methods." At the end
of the second exposure, the cells were released from the
blockade and monitored for DNA synthesis, Ml, and cellularity.
.«»Htl
i
l
T
4
DAYS IN CULTURE
Chart 2. Differentiation pattern of nonsynchronized HL-60 cells detected in
the 6 days after Me.-SO exposure. Differentiation is expressed as the percentage
of NBT-positive cells. Various curves refer to cultures incubated for different
periods with the inducer.
Table 1
Functional differential and morphological changes of nonsynchronized cells
exposed to Me2SO for 30 hr
Functional tests were performed as described in "Materials and Methods."
2,5-
Morphological differentiation was performed on Wright-Qiemsa-stained
slides. Results are means of 3 different experiments.
Functional differ
entiation (% of
cells)Time(days)after
'9
differentiation
counts)Myelo- tial
Cytospin
(differen
|i
2
blastsand
pro-myelo-cytes9594885959Myelo-cytes56122413Meta-m
8
re
in
cytosis442543129NET
duction35483329Morphological
duction01235Phago
0,5
1
2
3
DAYS IN CULTURE
Chart 1. Cell growth of nonsynchronized HL-60 cells after different exposure
periods to Me2SO. •¿,
6 nrs exposure; O, 30 hr exposure; A, 72 hr exposure;
A, 120 hr exposure.
446
" Differentiation
pattern analyzed during 5 days, with Day 0 as the first day of
exposure to Me2SO.
CANCER
RESEARCH
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VOL.
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Induction Pattern of HL-60 Cell Differentiation
[3HJThymidine incorporation
reached a peak in about 5.5 hr
(Chart 3a), and the Ml peak was reached at about 9 hr (Chart
3b). The second thymidine incorporation peak occurred at
about 16 to 18 hr and was broader.
The length of each phase was estimated according to the
method of Volpe and Volpe (29). In our experimental condi
tions, the average duration of S was 5.5 hr, that of G2 was 3 hr,
and that of M was 1.5 hr. GÌ
ranged between 7 and 8 hr, and
the total cell cycle time was about 16 to 18 hr; the second DNA
peak was too widely spread to permit its precise determination.
However, the sharp first peak of S phase and the subsequent
M phase clearly demonstrate the high rate of synchrony
achieved with the double block. Synchronization is further
supported by the doubling of cell density observed shortly after
the mitosis peak (Chart 3c).
No changes in the average phase duration were noted in
cells that proceeded through synchronized S, G2, M, and G,
phases in the presence of Me2SO, and cell viability remained
over 90%.
Effect of Me2SO on Synchronized HL-60 Cells. Synchro
nized cells were released from the thymidine block and resuspended into fresh medium containing 1.25% Me2SO. Cells
were maintained in the presence of the inducer for the same
periods of time assayed in nonsynchronized cells, i.e., 6, 12,
18, 24, 30, 44, 72, 96, and 120 hr.
Identical proliferation was observed in all cultures during the
first 2 days, and doubling was achieved in 10 to 12 hr. From
Day 3, growth appeared to depend on the time of exposure, as
observed in nonsynchronized cells. Phagocytosis activity, mor
phological changes, and NBT reduction ability showed a pat
tern of appearance similar to that of the nonsynchronized cells.
Thymidine alone induced differentiation in a low percentage
of cells (12 to 19% of NBT-positive cells), even in the absence
by Me2SO
of Me2SO. No change in the amount of differentiated cells was
observed when synchronized cells were incubated for 6 hr in
the presence of Me2SO, while a significant increase was de
tected after 12 hr of Me2SO exposure. Prolonging the time of
incubation with Me2SO increased the proportion of differen
tiated cells (Chart 4). For incubation times up to 30 hr, the
percentage of NBT-positive cells was slightly higher in syn
chronized than in nonsynchronized cells, whereas afterwards
the differences were undetectable. The linear rise in the per
centage of differentiated cells, observed in both synchronized
and nonsynchronized cells with increasing periods of Me2SO
exposure, is compared in Chart 5.
In order to determine if Me2SO promotes expression of
myeloid differentiation by an action related to a specific phase
of the cell cycle, synchronized cells were exposed to Me2SO
for 12 hr at different stages of the cell cycle: (a) immediately
after the release of the block (during S-G2 and M phases); (t>)
6 hr later (during G2-M and d phases); and (c) 12 hr later
(during d phase and the second S phase). The same amount
of differentiated cells were detected in each culture (Table 2),
indicating that the effect of Me2SO was not altered when
exerted at different phases of the cell cycle.
The action of Me2SO was then tested on cells blocked by a
double thymidine exposure. At the 10th hr of the second
thymidine exposure, Me.-SO was added and was maintained
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DAYS
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Chart 4. Differentiation pattern of synchronized HL-60 cells detected in the 6
days after Me..SO exposure. Differentiation is expressed as the percentage of
NBT-positive cells. Various curves refer to cultures incubated for different periods
with the inducer.
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Chart 3. Synchronization of HL-60 cells by 2 sequential exposures to 2 mw
thymidine. Cells were released from the thymidine block and monitored for DNA
synthesis expressed as [3H]thymidine uptake (a), Ml (b). and cell density (c).
FEBRUARY
1982
18
24
30
36
42
48
72
120
96
DMSO EXPOSURE [hours)
Chart 5. Relationship between exposure length to Me..SO (DMSO) and induc
tion to differentiation in synchronized (•)and nonsynchronized (•)HL-60 cells.
Cells were exposed to Mo. SO for periods varying from 6 up to 120 hr. Differen
tiation is expressed as the maximum percentage of NBT-positive cells obtained
for each given exposure time.
447
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C. Tarella et al.
Table 2
Effect of addition of Me2SO during different phases of the cell cycle
Time of exposure" (hr)
Cell cycle phases0
NBT-positive cells0 (%)
0-12
6-18
12-24
S-G2—M
Gî-M-G,
G,-S
30 ±4°
33 ±4
29 ±3
Cells synchronized by thymidine block were incubated for 12 hr with Me. SO
immediately after the block release (0 to 12). 6 hr later (6 to 18). and 12 hr later
(12 to 24).
b Cell cycle phases which cells exposed to Me2SO were going through.
c Differentiation expressed as maximum percentage of NBT-positive cells
detected at Day 2 of culture.
d Mean ±S.D. of 3 different experiments.
for 12 hr in the presence of the nucleoside. The number of
cells remained unchanged, and the Ml was always less than
0.2%. The cells were then washed and transferred into RPMI
1640-fetal calf serum alone. The subsequent differentiation
degree was significantly higher after the addition of Me2SO to
the cultures (6, 55, and 29% NBT-positive cells at the 24th,
48th, and 72nd hr, respectively). This result indicates that a
high induction to differentiation can be obtained in nonproliferating HL-60 cells treated for short periods with Me2SO.
In order to exclude that the commitment event occurs after
the thymidine block is released, by residual Me2SO that might
remain in the cells, some cultures were maintained in the
presence of thymidine for additional 12 hr, after ME2SO ex
posure and then transferred into RPMI 1640-fetal calf serum
alone. The time of appearance of differentiation markers was
not delayed by the prolonged thymidine incubation, as one
would expect if the action of Me2SO were exerted after the
release from the block. In fact, the percentages of NBT-positive
cells after the addition of Me2SO to the cultures were 10, 58,
and 30 at the 24th, 48th, and 72nd hr, respectively, indicating
that no more cells were committed to differentiation in 12 hr
after the Me2SO was removed.
DISCUSSION
The pattern of differentiation induced in cultured HL-60 cells
by Me: SO was determined using both morphological and func
tional criteria. A close correlation was found between the
percentage of cells induced to differentiate morphologically
beyond the promyelocyte stage and the percentage of func
tionally mature cells as described by Collins era/. (6). However,
the development of functional markers preceded the appear
ance of morphological changes, in agreement with the findings
of Newburger et al. (18).
A detectable increase in the percentage of differentiated
cells was observed only after 12 hr of continuous incubation in
the presence of Me2SO. Exposure for 12 hr is therefore the
minimal requirement to promote terminal myeloid differentiation
in a significant fraction of HL-60 cells. However, a latent period
was noted between induction by Me..SO and the appearance
of differentiated functions, since cells exposed for 12, 18, and
24 hr and then transferred into Me2SO-free medium showed
morphological and functional differentiation markers between
the 30th and the 72nd hr. Thus, 2 sequential events can be
distinguished in in vitro differentiation. They are transition from
an uncommitted to a committed state, which requires the
presence of Me2SO, and then the expression of biochemical
functions associated with a differentiated phenotype. This sec
ond event probably does not require Me2SO. Differentiation of
448
induced HL-60 cells, therefore, seems to be an irreversible and
stable process and continues even if the inducer is removed
from the medium. These findings are consistent with published
results in Friend erythroleukemia cells (10, 19).
Although a minimal exposure time of 12 hr was required for
significant induction of differentiation, the proportion of com
mitted cells was directly correlated to the length of exposure to
Me2SO, since an increasing number of cells was induced with
increasing periods of incubation with the inducer. This can be
explained by either a stochastic model for the commitment
process or a genetic heterogeneity of the cell population in its
sensitivity to Me2SO. Genetic heterogeneity seems unlikely to
explain the pattern of differentiation that we detected in Me2SOtreated HL-60 cells. We observed, in fact, that a small amount
of uninduced cells was still present in cultures exposed to
Me2SO for as long as 120 hr. These cells were able, if trans
ferred to Me2SO-free medium, to reconstitute a population of
self-renewing HL-60 cells, which, when rechallenged with
Me2SO, did not show a diminished sensitivity to the inducer, as
would be expected if genetic heterogeneity were the explana
tion.
We would suggest, then, that a stochastic model underlies
the behavior of cultured HL-60 cells. According to this model,
commitment events occur randomly in the population with a
particular probability. This probability reflects the likelihood
that a cell will undergo differentiation under given culture
conditions. A stochastic model has been suggested for the
commitment to differentiation of the pluripotent hematopoietic
stem cell (28). It has also been proposed for Me2SO-induced
differentiation of Friend erythroleukemia cells (9, 19, 22). In
addition, a stochastic model is consistent with the initiation of
eukaryotic cell replication process (26).
HL-60 cell differentiation could be explained by sensitivity of
the cells to the inducer only during a particular phase of the
cell division cycle. For instance, cultured cells could be induced
to differentiate only when they enter the active proliferating
state, and the decision to enter this state might be stochasti
cally controlled. To investigate whether this mechanism is true
for HL-60 cells, cells synchronized with a double thymidine
block were induced with Me2SO. In synchronized cells, the
percentage of differentiated cells for incubation times with
Me2SO up to 30 hr was slightly higher than nonsynchronized
cells. However, synchronized cells showed a response pattern
to Me2SO similar to that of nonsynchronized cells, in that a
direct relationship was observed between the percentage of
differentiated cells and the length of exposure. If the action of
Me2SO were related to a specific phase of the cell cycle, one
would expect a sharp increase in the amount of differentiated
cells each time the cells go through the sensitive phase in the
presence of the inducer. This was not observed in our experi
ments. Cells going synchronously through the cycle showed a
linear rise in the percentage of induction with increasing pe
riods of exposure. In addition, identical patterns of induction
were obtained in cultures exposed to Me2SO for 12 hr during
3 different periods of the cycle. Both results strongly support
the idea that the inducing effect of Me-SO is not linked to the
cell cycle. This hypothesis is further supported by the demon
stration that even nonproliferating cells blocked at the begin
ning of the S phase of the cell cycle can be induced to
differentiate by Me2SO. The higher rate of differentiation that
we detected in cultures treated with thymidine and Me2SO can
CANCER
RESEARCH
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VOL. 42
Induction Pattern of HL-60 Cell Differentiation
be attributed to a cumulative effect, since slight but significant
differentiation was observed with thymidine alone.
Our results are consistent with the finding reported by Rovera
et al. (23) that transition through the S phase is not necessary
for differentiation of 12-O-tetradecanoylphorbol-13-acetatetreated HL-60 cells into macrophage-like cells. The lack of
requirement of DNA synthesis for induction of myeloid differ
entiation of HL-60 cells is supported by several reports using
MEL cell lines (12,13), but not by others (8,10,15,17).
While
the reasons for these differences in the MEL system are not
clear, our experimental approaches using HL-60 cells had no
adverse effect on cell viability, cell cycle time, cytokinesis, or
the ability of the cells to commit to differentiation.
Therefore, a stochastic model not linked to the cell cycle is
proposed as the basis of induction of differentiation of HL-60
cells by Me2SO. Commitment to differentiation occurs in the
absence of DNA synthesis. Its molecular basis remains un
known. In Friend erythroleukemia cells, interactions of inducing
agents with cell membrane (1, 16), as well as with DNA (24,
27), have been reported. Since Me2SO and other inducing
agents can cause cells in every phase of the cell cycle to
differentiate irreversibly to a nonproliferating state, it is possible
that this system may be useful in studying antileukemic agents
which must act by inhibiting cell proliferation in some manner.
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1982 American Association for Cancer Research.
Induction of Differentiation of HL-60 Cells by Dimethyl
Sulfoxide: Evidence for a Stochastic Model Not Linked to the
Cell Division Cycle
Corrado Tarella, Dario Ferrero, Eugenio Gallo, et al.
Cancer Res 1982;42:445-449.
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