Inhibition of Human Leukocyte Mitosis
by Prednisolone in Vitro*
PETER C . NOWELL
(Department of Pathology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania)
SUMMARY
The water-soluble glucocorticoid, prednisolone ~l-phosphatc, has been shown to
inhibit mitotic activity of normal human leukocytes in short-term culture, with a
roughly linear relationship between the log dose of steroid and the percentage decrease
in mitosis. Over a concentration range of .002 ug/ml to 10 pg/ml, the mitotic index
decreased to 25 per cent of control values. Further studies indicate that most of this
inhibitory effect of the steroid operates during the first ~4 hours in tissue culture,
although the effect is not demonstrable until the 2d and 3d day, when mitosis begins.
The findings indicate that prednisolone does not significantly inhibit mitosis directly,
in these cultures, but rather inhibits the conversion of partially differentiated circulating leukocytes to a state capable of mitosis. This action occurs at the same time as
that of phytohemagglutinin, which initiates mitosis in these cultures by stimulating
the conversion process.
M A T E R I A L S AND M E T H O D S
Leukocytes were separated by means of phytohemagglutinin from the peripheral blood of healthy
donors and were grown in culture for 3 days according to the technic described by Moorhead et al.
(4). Each standard culture consisted of 10 million
cells in 8 ml. of medium (3 ml. autologous plasma,
5 ml. TC-199). Colchicine was added 5 hours prior
to termination of the cultures. The cells were harvested, pretreated, fixed, air-dried, and stained as
previously described (4). The mitotic index of each
culture was determined by counting the number of
metaphases in 2000 cells. Prednisolone 21-phosphate, 1 dissolved in distilled water, was added to
the cultures at the time of planting, with an equal
volume of water added to the controls. Final concentration of steroid in the cultures varied from
0.002 #g/ml to 10 #g/ml.
In some experiments, the following modifications of the technic were employed:
1. Standard cultures were terminated at 2 days
and at 4 days, instead of at 3 days, to test the effect of prednisolone (0.5 gg/ml) over these time
periods.
* This investigation was supported by Senior l~esearch Fcl2. Cultures were planted without prednisolone,
lowship SF-4 from the U.S. Public Health Service and by
1The prednisolone 21-phosphate was supplied by Dr.
Grant C-4659 from the National Cancer Institute of the NaRichard T. Smith, Merck, Sharp and Dolmae, West Point,
tional Institutes of Health, U.S. Public Health Service.
Pennsylvania. Its glucocorticoid activity is 4-5 times that of
Received for publication June 5, 1961.
hydrocortisone.
The involution of lymphoid tissues in response
to treatment with adrenal cortical steroids is apparently the result of a dual mechanism: death of
mature lymphocytes and inhibition of mitosis in
immature lymphocytes (3). The latter phenomenon, mitotic inhibition by cortisone and related
compounds, has been demonstrated both in vivo
and in vitro with various cell systems (8), but such
studies on lymphoid cells have not clearly demonstrated whether the steroid was operating by slowing down cells already moving through a mitotic
cycle, or by preventing activation of mitotically
inactive cells (see references in [3] and [8]).
A technic has recently been described in which
partially differentiated monocytes and large lymphocytes from normal human peripheral blood are
converted to a state capable of mitotic activity in
culture by the plant extract, phytohemagglutinin
(6). This system provides a means of investigating,
with normal human lymphoid cells, various aspects of mitotic inhibition by adrenal cortical hormones. For this purpose, the water-soluble glucocorticoid, prednisolone 21-phosphate, 1 was used.
1518
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NowELL---Inhibition of Leul;ocyle Mitosis by Prednisolone
had prednisolone added after 1 day or after ~ days,
and then were terminated, as usual, on the 8d.
These experiments tested the effect of having
prednisolone (0.5 /ag/ml and 10 ~g/ml) present
only for the last 24 or 48 hours of the culture period.
8. Cultures were made of leukocytes which had
been separated from whole blood without the use
of phytohemagglutinin (PHA). These PHA-free
cultures were treated with prednisolone (0.5 pg/
ml) at the time of planting, and then with P t t A 1
hour later. Control cultures for this experiment
were treated with PIIA first and then with prednisolone 1 hour later. All were terminated routinely
on the 8d day to determine the effect on mitotic activity of exposure to prednisolone before PtIA.
From five to fifteen cultures were tested under
each experimental condition, with a control culture of the same donor's leukocytes running concurrently in each ease. Mitotic indices in the control cultures ranged from ~1 to 138 metaphases per
1000 cells (mean = 57) in the 3- and 4-day cultures, and from 4 to ~6 per 1000 (mean = 10) in
the ~-day cultures. The low ~-day values are consistent with previous evidence (5, 6) that mitotic
activity in these peripheral blood cultures is minimal before the 3d day.
In a number of representative control cultures
and prednisolone-treated cultures (0.5 ~g/ml for
13 days and 0.5 t~g/ml for final ~4 hours), the total
number of cells remaining at the time of termination was determined, as well as the relative percentages of large mononuelear cells and small
lymphoeytes. In addition, small glass slides were
placed in these cultures (5, 6), and, after being
stained with Giemsa, these preparations were used
to compare cell morphology in the treated and
untreated cultures.
RESULTS
The control cultures, after 3 days, were similar
to those described previously (5, 6). Approximately half of the original inoculum remained, consisting of small lymphocytes (~5 per cent), large
mononuclear cells resembling blasts (75 per cent),
and an occasional granulocyte. The number of
cells and the percentage of small lymphocytes in
prednisolone-treated cultures was the same as in
the controls. IIowever, in the cultures exposed to
prednisolone for 3 days, fewer of the large mononuclear cells had the "blast" appearance characteristically associated with mitotic activity in
these cultures, and more had the "macrophage"
morphology typically associated with differentiation and degeneration (5).
The inhibitory effect on leukocyte mitosis, in
1519
standard 3-day cultures, of concentrations of prednisolone varying from .00~ #g/ml to 10 gg/ml is
demonstrated in Chart 1. There was a roughly
linear relationship between log dose of the steroid
and decrease in mitotic activity. The minimum
concentration, .00~ pg/ml, had no measurable effect, while the maximum concentration, 10 #g/ml,
reduced mitotic activity by nearly 75 per cent.
The result of delayed addition of prednisolone
to 3-day cultures is presented in Chart ~. Prednisolone treatment during only the last ~4 hours of
culture had no effect on mitosis at the 0.5 #g/ml
level, and only a slight effect at the 10 #g/ml level.
Prednisolone acting for the last 48 hours (i.e.,
added after 1 day of culture) had greater effect at
both the 0.5 #g/ml and 10 pg/ml levels than did
prcdnisolone acting for only the final ~4 hours, but
o
n..I-(D
N
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M
W
c~
z
V-
120
I00
60
40
20
r f lfl,,ll
N
.001
I i t,lllll
~ i iJ~rlll
0.1
1.0
I0.0
PREDNISOLONE (;ug/ml)
I i Ill,ill
901
CItART 1.--Effect of various concentrations of prednisolone
on mitosis in 3-day cultures of normal, h u m a n leukocytes.
Mitotic index in prednisolone-treated cultures is expressed as
per cent of mitotic index of control cultures, which varied from
'21 to 138 metaphases/1000 cells (mean - 57).
the effect was minor compared with that of similar
doses acting from the time the cultures were
planted.
The effect of prednisolone (0.5 ~g/ml) in standard cultures terminated at ~ days and at 4 days is
given in Chart 3. As shown, mitotic inhibition in
both instances was the same as in standard 3-day
cultures treated with the same concentration of
steroid.
The effect of adding prednisolone (0.5/zg/ml) to
cultures containing no phytohemagglutinin and
then adding phytohemagglutinin 1 hour later is
also shown in Chart 3. These cultures were terminated at 3 days and showed the same mitotic
inhibition as cultures which contained phytohemagglutinin before the addition of prednisolone.
DISCUSSION
The results indicate that the water-soluble glucocorticoid, prednisolone ~l-phosphate, inhibits
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1961 American Association for Cancer
Research.
Cancer Research
15~0
mitotic activity in primary cultures of normal human leukocytes. A concentration of prednisolone
(0.02 tzg/ml) comparable in glucocorticoid activity
to normal levels of free cortisol in human plasma
(0.08 tzg//ml) (3), caused approximately 20 per
cent reduction in mitotic activity in the leukocyte
cultures, and greater concentrations produced a
further decrease. The roughly linear relationship
between log dose of prednisolonc and per cent decrease in mitosis is consistent with previous studies
of mitotic inhibition (7) and other physiological efPREDNISOLONE O. 5 ~ g / m l
PREDNISOLONE IO~ug/ml
I00
,J
o
~ B0
o
Vol. ~1, D e c e m b e r 1961
mitotic cycle, but rather is preventing the conversion of quiescent cells to a mitotically active state.
It is also during this first 24 hours in culture
t h a t phytohemagglutinin, the plant extract which
initiates mitosis in these cultures, seems to act (6).
Prednisolone apparently inhibits the conversion
process which phytohemagglutinin stimulates.
There does not, however, appear to be direct antagonism between the two compounds (i.e., competition for the same binding site), since the action of
the steroid was the same whcther the leukocytcs
were exposed to it shortly before or shortly after
the addition of phytohemagglutinin. Nor does the
effect of prednisolonc seem to be simply a brief
delay of the conversion process initiated by phytohcmagglutinin, since steroid-treated cultures examined at 4 days showed the sanle decrease in
mitotic activity as those terminated at 3 days.
I00
o 60
.J
o
n,-
X
N e0
~ 40
PREDNISOLONE
O. 5Jug/ml
o
I.L
o 6O
~ 20
I.--
x
N~
0
z
F U ~ L FINAL F N A L
FULL F NAL F I N A L
72
24
48
72
24
48
HOURS OF EXPOSURE TO PREDNISOLONE
CHART o..--Effect of delayed addition of prednisolone (0.5
~g/ml or 10 pg/ml) to standard 3-day cultures. Mitotic activity in cultures containing prednisolone for only the final ~4 or
48 hours is compared with that in cultures containing prednisolone from the time of planting (7~ hours). Each value is the
mean of at least five determinations.
fects of steroids. The action of prednisolone appears to involve specifically the cell division mechanisms, since there was no evidence of increased
cell destruction in the cultures.
The present findings further indicate that prednisolone exerts most of its inhibitory effect during
the 1st day in culture. Prednisolone added after
the first 24 hours had only minor effects, even at
the 10 ug/ml level, and, since 2-day cultures
treated with prednisolone from the outset showed
mitotic inhibition, the relative lack of inhibition in
3-day cultures treated only over the last 48 hours
was not due simply to insufficient exposure time.
During the first 24-36 hours in culture, the
monocytes and large lymphocytes in the inoculum
are changing over to a mitotically active state.
Synthesis of D N A and mitotic activity in the cultures is minimal during this period (5, 6). Thus, it
appears that prednisolone, acting during the first
24 hours, is not inhibiting cells moving through a
o20
i
:3- DAY
2-DAY
4-DAY
3-DAY
CULTURES CULTURES CULTURES CULTURES
(PREDNISOLONE
(STANDARD)
ADDED BEFORE
PHYTOHEMAGGLUTININ)
Cr~ART 3.--Effect of prednisolone (0.5 izg/ml) in standard
3-day cultures corrlpared with the effect in standard cultures
terminated at ~ days and at 4 days, and with the effect in cultures to which prednisolone was added 1 hour before phytohemagglutinin. Each value is the mean of at least five determinations. Mitotic indices in 3- and 4-day control cultures
varied from ~1 to 138 metaphases/1000 cells (mean = 57); in
~-day control cultures, from 4 to ~26 metaphases/1000 cells
(mean = 10).
t[ow prednisolone acts to produce this inhibitory effect is not clear. It has been postulated t h a t
mitotic inhibition by cortisone and related compounds results from alterations in glucose metabolism (1), but such studies have involved cells already mitotically active, exposed to steroid for
only a few hours. Swann (8) has suggested t h a t
such a phenomenon is fundamentally different
from the effect of a hormone on the conversion of
partially differentiated cells to a mitotically active
state, the situation which seems to be present here.
In this regard it is of interest that, in our culture
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Research.
N owELL--Inhibition of Leukocyte Mito~gs by Prednisolone
system, t r e a t m e n t with prednisolone p h o s p h a t e of
leukocytes a l r e a d y m i t o t i c a l l y active a t t h e time
of p l a n t i n g (i.e., r a t bone m a r r o w , h u m a n leukemic cells) has, t h u s far, failed to produce a n y consistent effect on mitosis3
N o t only is t h e m e t a b o l i c action of t h e glucocorticoids still in dispute, b u t also their site of action in t h e cell. T h e r e is r e c e n t evidence t h a t prednisolone m a y act on t h e cell m e m b r a n e r a t h e r t h a n
within the cell [see references in (~)]. T h i s is of particular interest, since p h y t o h e m a g g l u t i n i n also
m a y a c t a t the cell surface (6). Obviously, f u r t h e r
investigation is indicated. A l t h o u g h t h e p r e s e n t
culture s y s t e m is a n artificial one, in t h e sense t h a t
mitosis is initiated b y a nonphysiological m e c h a nism, p h y t o h e m a g g l u t i n i n , it a p p e a r s t h a t these
cultures can be used to explore basic questions of
cell division a n d h o r m o n e action on cells. Ultim a t e l y , it m a y bc possible to relate such studies to
the still u n d i s c o v e r e d m e c h a n i s m s controlling leuk o c y t e p r o d u c t i o n in t h e b o d y .
ACKNOWLEDGMENTS
The co-operation of the staff of the Blood Donor Station,
Ilospital of the University of Pennsylvania, and the able tcchUnpublished data.
15~1
nical assistance of Elizabeth Krohnert and Sandra Ferry are
gratefully acknowledged.
REFERENCES
1. BULLOUG~, W. S. Hormones and Mitotic Activity. Vit. &
Itorm., 13:261-92, 1955.
2. CItmSTENSEN, II. N. Action of Corfisol on Trapping of
Amino Acids by the Liver. In: G. E. W. WOLSTEN~O~XE
and M. O'CoN~'OR (eds.), Metabolic Effects of Adrenal
IIormoncs, pp. 56-64. Boston: Little, Brown & Co., 1960.
3. DOUGIIERTY,T. F. Adrenal Cortical Control of Lymphatic
Tissue Mass. In: F. STOULM~N(ed.), The Kinetics of Ccllular Proliferation, pp. 264-74. New York: Grune & Stratton,
1959.
4. MOORItEAO, P. S.; NOWELL, P. C.; MELLMAN, W. J.;
BATIPI'S, D. M.; and HUNGERFORD, ]). A. Chromosome
5.
6.
7.
8.
Preparations of Leukocytes Cultured from Human Peripheral Blood. Exp. Cell Research, 20:613-16, 1960.
NOWELL, P. C. Differentiation of IIuman Leukemic Leukocytes in Tissue Culture. Exp. Cell Research, 19:267-77,
1960.
- - - - . Phytohemagglutinin: An Initiator of Mitosis in Cultures of Normal Human Leukocytes. Cancer Research, 20:
46~-66, 1960.
PFEIFFER, E. F.; SANDRITTER, W., and SCHOFFLI~a, K.
Thymusmitosenhcmmung als quantitativer Nachweis der
Ncbennierenrindenaktivierung in Tierexperiment. Klin.
Wchnschr., 43:1028-~5, 195~2.
SWANS,M. M. The Control of Cell Division: A Review. II.
Special Mechanisms. Cancer Research, 18:1118-60, 1958.
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Research.
Inhibition of Human Leukocyte Mitosis by Prednisolone in
Vitro
Peter C. Nowell
Cancer Res 1961;21:1518-1521.
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