Published Online: 1 April, 1966 | Supp Info: http://doi.org/10.1083/jcb.29.1.

1
Downloaded from jcb.rupress.org on April 23, 2019

S T U D I E S ON T H E M E C H A N I S M OF H O R M O N A L
I N D U C T I O N OF A L K A L I N E
PHOSPHATASE IN HUMAN CELL CULTURES

I. Effects of Puromycin and Actinomycin D

MARTIN J. G R I F F I N and R O D Y P. C O X

From the Department of Medicine, New York University School of Medicine, New York, and The
Summer Research Institute of the Will Rogers Hospital, Saranae Lake, New York. Dr. Griffin's
present address is the Department of Cancer Research, University of Oklahoma School of Medicine,
Oklahoma City

ABSTRACT
Increased alkaline phosphatase activity is induced in certain epithelial cell cultures by
hormones with adrenal glucocorticoid activity or their analogues such as prednisolone
(ALhydrocortisone). Enzyme induction occurs in two distinct phases. During the first 12
hr after the addition of prednisolone, there is a small increase in alkaline phosphatase
levels. After 15 to 24 hr, the enzyme activity shows a sudden, marked linear rise, reaching
a maximum at 60 to 80 hr. Puromycin blocks enzyme induction immediately, even when
added during the period of rapid increase of enzyme. Actinomycin D blocks induction when
added no later than 8 hr after the addition of prednisolone. On the other hand, Actinomycin
D added during the phase of rapid enzyme induction has no effect for at least 12 hr. These
findings suggest that de novo protein synthesis is involved in prednisolone induction of
alkaline phosphatase and that the RNA messenger for this enzyme is relatively stable.

INTRODUCTION
Regulation of cell function in higher animals is
controlled in part by hormones. The mechanisms
of hormone action are probably complex and
include effects on cell permeability, alterations in
subcellular compartmentation, as well as changes
in the activity of certain enzymes. It is likely that
hormonal regulation of enzyme activity depends
not only on the particular hormone but also on
the enzyme system and its localization in the cell.
Recent studies suggest several mechanisms
whereby hormones may influence enzyme levels
in cells.
Steroid hormones may influence the substrate
specificity of certain enzymes (e.g., conversion of
glutamic dehydrogenase to alanine dehydrogenase)
by producing configurational changes in the
enzyme (1). Enzyme synthesis in cells also may be
regulated by hormones. Recent evidence derived
from studies on several different enzyme systems
such as tyrosine-a-ketoglutarate and glulamic-
alanine transaminases and tryptophan pyrrolase
indicate that hydrocortisone promotes de novo
synthesis of these enzymes, while substrates
promote an increase in the activity of these
enzymes by protecting them from cellular degrada-
tion (2-6).
Alkaline phosphatase is induced by adrenal
glucocortcoids in human leukocytes in vivo (7)
and, during embryonic development, in amphibian
(8), chicken (9, 10) and mouse intestinal epi-
thelium (11). In certain h u m a n epithelial cell
lines in tissue culture, the enzyme can also be
induced by hydrocortisone or its analogue pred-
nisolone (12, 13). T h e s e dispersed cell cultures of
either the monolayer or suspension type have
no a p p a r e n t intercellular orientation and are
composed of a relatively homogeneous cell popu-
lation. Therefore, they have the advantage of being
relatively simple cell systems for the study of hor-
m o n e action and enzyme induction.
T h e aims of the present study were: (a) to
determine the effect of prednisolone on the
kinetics of synthesis of alkaline phosphatase in
suspension culture; (b) to examine the effects of
certain inhibitors of both R N A and protein
synthesis on induction of the enzyme in order to
distinguish between its de novo synthesis and its
protection from cellular degradation; (c) to
estimate in a relatively simple cell system the
m i n i m u m stability of the messenger R N A for
alkaline phosphatase; and (d) to compare the
effects of an adrenal glucocorticoid analogue on
cell multiplication in monolayer cultures and in
suspension cultures.
MATERIALS AND METHODS
1. Cells and Media
Suspension cultures of HeLa S3 cells were grown
in Eagle's minimal medium (MEM) without calcium
ions (14). These cultures were furnished by Dr.
Matthew Scharf of Albert Einstein College of Medi-
cine. The methods of suspension culture were similar
to those described by McLimans et al. (15). For
kinetic experiments, 10-ml aliquots of the cell su-
spension were removed at various times from the
spinner flasks. The volume of medium was main-
tained by replacing the aliquots with fresh medium
containing the same concentrations of compounds.
This HeLa $3 cell line was also adapted to monolayer
culture and was studied while growing in Eagle's
minimal essential medium (MEM) (14) and in
Waymouth's medium (16), both containing 10%
calf serum. Another HeLa S~ cell line, obtained from
Dr. Harold Nitowsky of Johns Hopkins School of
Medicine, was grown in monolayer culture using
Waymouth's medium containing 10% human
serum. The methods of monolayer culture and the
techniques of analysis used in this laboratory have
been described (13, 17). Experiments using mono-
layer cultures were done in triplicate. All of the cell
lines used were free from pleuropneumonia-like
organism [PPLO] infection as determined by Dr.
Morton Davidson.
2 TirE JOURNAL OF CELL BIOLOGY • VOLUME ~9, 1966
2. Chemical Determinations
Cells were prepared for enzyme assay and protein
determinations as previously described (13, 17).
Alkaline phosphatase activity of cell lysates was
measured spectrophotometrically with p-nitrophenyl
phosphate (8 mM) as substrate in 0.5 ~ 2-amino-2-
methyl-l-propanol (AMP) buffer, pH 10.6. Specific
activity was expressed as #moles of p-nitrophenol
liberated in 15 rain per mg of protein (18). Acid
phosphatase was determined by the method of Lowry
(18). Protein was assayed by Lowry's method (19).
3. Radioactive Isotope Incorporation
Studies
In certain experiments, cultures were grown in
medium containing either leucine-l-Cn or uridine-
2-C 14. Two methods were used to measure radioac-
tive leucine or nridine which has been incorporated
into cell protein and RNA, respectively.
MONOLAYER CULTURES : Cells grown in mono-
layer were harvested and the cell suspension was
washed twice in isotonic saline. The cells were then
suspended at 4°C in 5% trichloroacetic acid (TCA).
The ceil residue was collected on Millipore HA filter
and thoroughly washed with ice-cold 5% TCA.
The dried filters were placed on planchets and radio-
active measurements were made in a Nuclear Chi-
cago micromil window counter.
SUSPENSION CULTURES: Cells grown insuspen-
sion culture in medium containing radioactive iso-
topes were lysed with deoxycholate, and 0.2 ml of
lysate was mixed with 3.0 ml of ice-cold 1 N per-
chloric acid. The precipitate was sedimented at
1,600 Rv~ in an International Refrigerated Centri-
fuge at 0°C. The sediment was dissolved in 0.3 ml
of 1 N NaOH, and 15 ml of dioxane scintillation
solution were added (20). The sample was cooled
at 4°C in a polyethylene vial and then assayed for
radioactivity in a Packard Tricarb scintillation
counter. The background radioactivity was 45 CPM
and the counting efficiency was approximately 80%
for C 14.
4. Cytology
Mitotic indices were determined on cells grown in
suspension culture. The cell suspension was washed
in isotonic saline and the cells were air dried on
covcrslips. Following fixation in 95% ethanol for
10 to 15 rain, the coverslips were stained by the acetic-
orcein method. The mitotic indices were determined
by counting a minimum of 1500 nuclei per slide.
To determine alkaline phosphatase histochemi-
cally, cover slip preparations of HeLa $3 cells grown
in suspension culture were prepared as described
above. Alkaline phosphatase was determined histo-
chemically using Kaplow's modification of the Men-
ten, Junge, Green method (21) or by the postcoupling
technique of Burstone (22). Both methods gave
similar results.
RESULTS
Kinetics of Prednisolone Induction of Alkaline
Phosphatase in HeLa $3 Suspension Culture
Fig. 1 shows the kinetics of alkaline phosphatase
induction by prednisolone in HeLa $3 suspension
cultures. A representative experiment illustrates
three aspects of the induction curve as shown in
Fig. 1. During the first 18 hr of growth in medium
containing prednisolone, there is a small increase
in the alkaline phosphatase activity up to twice
the level observed in replicate cultures grown in
medium without the hormone (controls). There-
after, the rate of alkaline phosphatase induction
rises markedly and enzyme activity increases
linearly, reaching a value about 10-fold greater
than that of control cultures after 60 hr. The
rate of induction between 24 and 60 hr is 3.5
times faster than that seen during the initial 24 hr.
The plateau reached after 60 hr reflects the
maximal enzyme activity induced by prednisolone
under the conditions of this experiment. When
prednisolone-induced cultures are resuspended in
medium without prednisolone, enzyme levels
decrease at a rate which is consistent with dilution
by cell muhiplication. The relative alkaline
phosphatase activity of individual HeLa $3 cells
during prednisolone induction and alter resus-
pending in medium without prednisolone was
determined semiquantitatively by histochemical
methods. Coverslip preparations were incubated
with substrate until a faint staining of cells was
observed; the length of incubation and the intensity
of staining of individual cells correlate approxi-
mately with their enzyme activity. Coverslip
preparations made from suspension cultures
grown in medium containing prednisolone show
that the increase in enzyme activity is approxi-
mately the same throughout the cell population.
On removing prednisolone from the medium, the
enzyme activity appears to decrease propor-
tionately in all the cells. Acid phosphatase specific
activity remains unchanged during the experi-
ment.
Monolayer cultures of HeLa Sa cells in which
the enzyme is induced by prednisolone show
results similar to those described above (13). The
main difference between monolayer and sus-
pension cultures is the magnitude of enzyme
induction. During the first 15 hr, suspension
cultures show up to a 100% increase in alkaline
phosphatase levels. However, most of the cell
lines studied in monolayer cultures show an
increase in enzyme activity of only 10 to 20%
during this time (13). The enzyme activity in-
duced by prednisolone in monolayer cultures is
2- to 8-fold higher than control cultures, while
suspension cultures show an 8- to 20-fold increase.

Prednisolone removed
+ o.~2 l
< 0.18
I-
<[

a. 0.14
0
-I-
" 0.10
LU

..J
<[ 0.02 _ _ _ ¢ • : -
l | I | I I I I I "
24 4B 72 96 120 144

HOURS

FIUURE l Induction of alkaline phosphatase activity by 1.0 #g prednisolone in suspension cultures of

HeLa S~ cells. • Cultures grown in Eagle's spinner medium containing 7% of calf serum. O Replicate
culture grown in Eagle's spinner medium containing 7 ~ calf serum and prednisolone at 1.0 #g/ml. At 69
hr the cells grown in medium with prednisolone were suspended in medium without prednisolone, and
the decline in alkaline phosphatase specific activity was followed. -t-' Specific activity expressed as the
~moles of p-nitrophenol liberated in 30 rain at 87°C by deoxyeholate cell lysate containing 0.1 mg of pro-
tein.

M. J. GRIFFINAND R. P. Cox Mechanismof Hormonal Induction. 1 3

Effect of Prednisolone on Cell Multiplication
and Mitotic Index
In suspension cultures, prednisolone reduces
cell multiplication and increases the mitotic index
as in the case of cells grown in monolayer cultures
(13). There was no apparent change, during the
first 48 hr, in the generation time of 25 4- 5 hr in
suspension cultures grown in medium containing
prednisolone. However, at about 60 hr, the
generation time is increased to 45 4- 5 hr and the
mitotic index of 8 to 9 % is twice that of replicate
cultures grown in the absence of hormone. When
prcdnisolone-induced cultures are resuspcnded in
m e d i u m without prcdnisolone within 8 to 16 hr,
the generation time returns to 25 4- 5 hr.
Effects of Puromycin and Actinomycin D on
Prednisolone Induction of Alkaline
Phosphatase
HELA S3 MONOLAYER CULTURES GROWN IN
WAYMOUTH'S M E D I U M : For the first 12 to
18 hr of growth in medium with prednisolone,
there is little or no increase in alkaline phosphatase
(13). During the following 24 to 36 hr, the enzyme
levels increase rapidly. Therefore, in the experi-
ments described in Table I, puromycin was added
18 to 20 hr after adding prednisolone so that the
effects of the inhibitor could be observed during
the phase of rapid enzyme increase. The cultures
were harvested 24 hr after the addition of puro-
mycin.
Table I shows that puromycin at 0.1 #g per ml
did not affect either alkaline phosphatase induction
or leucine-C 14 incorporation. At a higher concen-
tration of puromycin, 0.5 #g per ml, the pred-
nisolone-induced increase in alkaline phosphatase
and the incorporation of leucine-C 14 into protein
were markedly reduced. The effect of puromycin
on alkaline phosphatase induction depends not
only on the concentration of the inhibitor but also
on the confluency of the monolayer. As shown in
Table I, recently transferred cultures containing
about 1 million cells when grown in the presence
of puromycin at 0.5 #g per ml showed evidence of
cell injury within 12 to 14 hr. In these cultures,
the alkaline phosphatase was decreased by one-
half while the acid phosphatase was markedly
reduced. Acid phosphatase, a constitutive enzyme
in cell cultures, is a sensitive indicator of cell
damage. Radioactive leucine incorporation was
reduced by 70 to 9 0 % in these cultures. O n the
4 THE JOURNALOF CELL BIOLOGY • VOLUME ~9, 1966
other hand, confluent 72-hr-old cultures con-
taining 4 to 5 million cells appeared cytologically
normal for the first 24 hr after adding puromycin,
and radioactive leucine incorporation was reduced
by only 35% of the control value. Hormonal
induction of alkaline phosphatase was markedly
reduced, however.
Cultures grown in medium containing both
puromycin and prednisolone showed less morpho-
logical damage as compared to replicate cultures
grown in medium with puromycin alone. The
steroid hormone appears to protect cells from the
cytotoxic effects of puromycin without interfering
with the inhibition of protein synthesis by the
drug.
Table II shows the effects of two concentrations
(0.025 #g per ml and 0.10 t~g per ml) of Actino-
mycin D on prednisolone induction of alkaline
phosphatase in H e L a $8 monolayer cultures.
The Actinomycin D at the lower concentration
did not affect alkaline phosphatase induction,
even when added at the same time as prednisolone.
This concentration of Actinomycin D reduced the
incorporation of phosphate-P s2 into cellular
macromolecules such as nucleic acids by 35%.
Increasing the Actinomycin D concentration to
0.10 ~g per ml Mocked hormonal induction of
alkaline phosphatase and reduced phosphate-P 32
incorporation by 68 %.
Table I I I shows that Actinomycin D at the
concentration which blocks induction when added
at the same time as prednisolone is without effect
when added 18 hr after the hormone.
HELA S3 CELL SUSPENSION CULTURES:
Fig. 2 shows the effect of adding either puromycin
at 5.0 #g per ml or Actinomycin D at 5.0 #g per
ml to suspension cultures during the phase of
rapid linear synthesis of the enzyme. When puro-
mycin, an inhibitor of protein synthesis, is added
to the cultures, prednisolone induction of alkaline
phosphatase promptly ceases and the specific
enzyme activity falls. The experimental results
have been plotted for only the first 12 hr after
adding puromycin. This concentration of puro-
mycin irreversibly damages cells within 7 to 10
hr. After 12 hr, the cells show altered microscopic
appearance with increased cytoplasmic granularity
and ballooning, as well as the presence of cell
fragments and debris. These cells have lost the
ability to exclude a 0.15 % solution of trypan blue.
Table IV shows the effect of puromycin at 5.0
#g per ml on the leucine-C I4 incorporation into
 TABLE I
Effect of Puromycin on Prednisolone Induction of Alkaline Phosphatase in HeLa Sa Monolayer Cultures

Puro- Leucine-
mycin 1-C14
Age of HeLa cultures before concen- Alkaline Acid incorpo- Appearanceof
adding prednisolone* tration* phoaphatase:~ phosphatase:~ ration monolayer

#g/ml Mean Range Mean Range units§

24 h r :
Control -- 0.07 0.09-0.06 0.039 0.043-0.031 10.4 Normal
P r e d n i s o l o n e 0.5 ttg/ml -- 0.24 0.28-0.22 0.044 0.051-0.038 9.5 Normal
Control 0.1 0.10 0.11-0.09 0.045 0.050-0.039 11.1 Normal
P r e d n i s o l o n e 0.5 ttg/ml 0.1 0.23 0.26-0.22 0.047 0.053-0.041 9.6 Normal

24 h r :
Control -- 0.06 0.07-0.04 0.012 0.016-0.009 12.0 N o r m a l
P r e d n i s o l o n e 0.5 # g / m l -- 0.16 0.18-0.15 0.014 0.016-0.010 11.5 N o r m a l
Control 0.5 0.03 0.03-0.02 0.001 -- 1.05 M a r k e d cell de-
generation and
detachment
P r e d n i s o l o n e 0.5 ~tg/rnl 0.5 0.03 0.04-0.03 0.001 1.30 M o d e r a t e d cell
degeneration
and detachment
72 h r :
Control -- 0.18 0.20-0.15 0.051 0.057-0.044 12.6 Normal
P r e d n i s o l o n e 0.5/~g/ml -- 0.75 0.83-0.71 0.050 0.056-0.046 15.0 Normal
Control 0.5 0.16 0.17-0.14 0.050 0.058-0.044 8.0 Slight cell re-
traction
P r e d n i s o l o n e 0.5 # g / m l 0.5 0.29 0.31-0.26 0.048 0.052-0.043 9.8 Normal

* P u r o m y c i n was a d d e d 18 to 20 h r after a d d i n g prednisolone, a n d c u l t u r e s were h a r v e s t e d 24 h r later.

/~moles p - n i t r o p h e n o l released/15 m i n / m g cell protein. E x p e r i m e n t s w e r e c a r r i e d o u t in triplicate; the
m e a n a n d r a n g e of specific activities are s h o w n .
§ CPM X 103/0.1 m g cell p r o t e i n .

TABLE II
Effect of Simultaneously Adding Actinomycin D and Prednisolone to HeLa $3 Monolayer Cultures
Actinomycin
D concentra- Alkaline Acid p32 incor-
Replicate HeLa $8 monolayer cultures tion phosphatase* phosphatase* poration~:

lsg/ml Mean Range Mean Range

Control -- 0.035 0.041-0.028 0.026 0.029-0.024 8.5
P r e d n i s o l o n e 0.5/~g/ml -- 0.178 0.196~0.154 0.024 0.027-0.020 8.4

Control 0.025 0.045 0.048-0.043 0.022 0.024-0.019 5.9

Prednisolone 0.5 ~tg/ml 0.025 0.260 0.300-0.220 0.024 0.025-0.023 5.4

Control -- 0.051 0.055-0.047 0.032 0.036-0.028 33.0

P r e d n i s o l o n e 0.5 ttg/ml -- 0.150 0.173-0.136 0.032 0.035-0.028 37.0

Control 0.100 0.051 0.056--0.048 0.027 0.031-0.025 5.7

P r e d n i s o l o n e 0.5 ~tg/ml 0.100 0.048 0.055-0.045 0.035 0.040-0.028 10.8

* ~tmoles p - n i t r o p h e n o l released/15 m i n / m g cell p r o t e i n . E x p e r i m e n t s were c a r r i e d out in triplicate; the

m e a n a n d r a n g e o f specific activities are s h o w n .
:~ cPM X 103/0.1 m g cell p r o t e i n .

M. J. GRIFFIN AND R. P. C o x Mechanism of Hormonal Induction. I 5

 TABLE III
Effect of Adding Actinomycin D at Various Times on Prednisolone Induction of Alkaline Phosphatase

"rime of
adding
Actinomycin Alkaline Acid p3~inccr-
Replicate HeLa S, monolayercultures D 0. I ~g/ml phosphatase* phosphatase* poration~t

hr Mean Range Mean Range

Control -- 0.011 0.0124).009 0.025 0.0274).023 9.5
Prednisolone 0.5 t~g/ml -- 0.050 0.054-0.044 0.030 0.031-0.028 10.8

Co ntrol 0 0.014 0.0164). 009 0.030 0.035-0.027 2.5

Prednisolone 0.5 ug/ml 0 0.013 0.0184).010 0.025 0.0284).022 2.3

Control 18 0.012 0.0144).010 0.027 0.0314) .024 5.2

Prednisolone 0.5 ug/ml 18 0.051 0.053-0.048 0.030 0.0344).025 6.0

* umoles p-nitrophenol released/15 m i n / m g cell protein. Experiments were carried out in triplicate;
the mean and range of specific activities are shown.
:~ oPM X 10~/0.1 mg cell protein.

0.15

1a.I
r,D
I-
<
-1-
n 0.10
1,0
o
¢1
la.I
z
._1
,,4 0.05
_J

HOURS

FIGURE ~ Effect of puromycin and Actinomycin D oil the iuduction of alkaline phosphatase by predniso-
lone in suspension cultures of HeLa S~ cells. All cultures were grown in Eagle's spinner medium contain-
ing 7% calf sermn. • Control culture--no additions. O Prednisolone 1.0 #g/ml added at time 0. A
Prednisolone 1.0 #g/ml added at time 0. 39 hr later, Actinomycin D 5.0 #g/ml was added. >< Predniso-
10ne 1.0 #g/ml added at time 0. 39 hr later, puromycin 5.0 gg/ml was added. *Alkaline phosphatase
activity--see legend to Fig. l.

protein. Radioactive leucine incorporation was alkaline phosphatase. Actinomycin D a d d e d

reduced by 6 3 % after 8 hr in the presence of
puromycin, and further induction of alkaline
phosphatase was completely blocked.
Fig. 2 also shows the effects of Actinomycin D
at 5.0 /~g per ml on prednisolone induction of
39 hr after prednisolone reduced uridine-C 14 in-
c o r p o r a t i o n into nucleic acids by 9 5 % (Table
IV). However, the rate of alkaline phosphatase
synthesis and the increase in total cell protein were
unaffected during the following 10 to 12 hr (Fig.

6 T H E JOURNAL OF CELL BIOLOGY . VOLUME ~9, 1966

 T A B L E IV
Effect of Actinomycin D and Puromycin on the Incorporation of Radioactive Compounds into R N A and Protein of
Prednisolone-Induced HeLa $3 Suspension Cultures*

Time after adding Control§ Puromycin§ Control§ Actinomycin§

inhibitor C14 compound:~ 5.0/~g/inl 5.0/*g/ml

hr
C-1-C 14-1eucine 2.5 0.3
8 " 7.6 2.8
12 " 8.5 3.3 m

~ 2-CX4-uridine m 0.5 0.3

8 ~ 55.0 2.7
12 " 45.2 2.0

* Inhibitors added 39 hr after adding prednisolone.

:~ Each radioactive compound added at a concentration of 10 mucurie/ml.
§ cP~ X 103/0.1 mg cell protein.

2). Beyond 12 hr, the data are unreliable because
of irreversible cell damage.
T h e effects of a low concentration of Actino-
mycin D (0,05 /zg per ml) were also studied at 8
hr and at 39 hr after adding prednisolone to
suspension cultures of H e L a $3 cells. This concen-
tration after 12 hr reduced radioactive uridine
incorporation into the nucleic acids of H e L a
cell suspension cultures by only 10 to 50%.
Alkaline phosphatase induction and increase in
total cell protein were not affected for at least
12 hr. However, morphological evidence of injury
similar to that observed with the high concen-
tration of actinomycin (5.0 #g per ml) was seen
within 12 to 16 hr after adding the inhibitor.
DISCUSSION
T h e kinetics of prcdnisolone induction of alkaline
phosphatase in human cell cultures differs somc-
what from the kinetics of enzyme formation in
bacteria after adding inducer. For example, during
~-galactosidase induction in E. coli, enzyme syn-
thesis begins within 3 min after adding inducer,
and the rate of increase of enzyme is lincar (24).
O n the other hand, in H e L a cells there appear to
be two different rates of alkaline phosphatase
synthesis which occur in sequence: an initial slow
rate of increase of enzyme is followed from 15 to
24 hr later by an abrupt, rapid linear synthesis.
Similar biphasic enzyme induction after addition
of hormone has also been observed in plant seeds
(25). The rapid phase of gibbcrcllic acid induction
of a-amylase in barley endosperm is preceded by
a 9- to 15-hr slow phase.
The data presented in this study strongly suggest
that de novo protein synthesis is involved in the
induction of alkaline phosphatase by prednisolone.
Puromycin, an inhibitor of the final steps of
protein synthesis, immediately blocks further
prednisolone induction of enzyme when added to
cultures during the rapid phase of enzyme increase.
In contrast, Actinomycin D (5.0/zg per ml) added
during the rapid phase of enzyme induction does
not interfere with enzyme synthesis for at least 12
hr following its addition to suspension cultures.
This concentration of inhibitor is known to act
within an hour (26) and reduce by 9 5 % the
incorporation of radioactive label into R N A .
This result is expected from studies on other
biological systems which have shown that Actino-
mycin D markedly inhibits DNA-dependent
R N A synthesis and to a lesser degree the synthesis
of protein (27, 28). Thus, previously formed
messenger R N A will be insensitive to the effects
of Actinomycin D and will be capable of directing
further protein synthesis until it is degraded. The
stability of this messenger R N A can be approxi-
mated as at least 10 to 12 hr, since Actinomycin D
at a concentration that completely inhibits new
R N A synthesis does not interfere with induction
for this period of time. The lifetime of certain other
R N A messengers in m a m m a l i a n cells is known to
be long (29, 30).
An intriguing aspect of the current studies is
the effect of prednisolone on the generation time
and mitotic index of H e L a Ss cell cultures. These
cells exhibit an increased generation time with
increased cytoplasmic growth and doubling of the
mitotic index. There appear to be a dispropor-
tionate number of these dividing cells in meta-

M. J. GRIFFIN AND R. P. Cox Mechanism of Hormonal Induction. I 7

phase. These effects are reversed within a few
hours of resuspending the cells in medium without
prednisolone. Semiquantitative estimation of
alkaline phosphatase activity by histochemical
methods shows that ceils in mitosis appeared to
stain slightly more intensely than cells in inter-
phase. However, all cells from cultures grown in
medium with prednisolone showed a marked
increase in alkaline phosphatase staining when
compared to cells grown in medium without the
hormone.
Prednisolone protects cells from the cytotoxic
effects of both puromycin and Actinomycin D
without affecting the inhibition of protein and
R N A syntheses. The mitigation by prednisolone
of this cellular injury may be similar to the 24-hr
delay in the cytopathic effects of poliovirus in-
fection observed in H e L a cell cultures grown in
medium with prednisolone (13). Adrenal gluco-
corticoids are also known to have a profound
REFERENCES
1. TOMKIN, G. M., and YEILDING, K. L., Regula-
tion of the enzymic activity of glutamic dehy-
drogenase mediated by changes in its structure,
Cold Spring Harbor Syrup. Quant. Biol., 1961,
26, 331.
2. KEN•EY, F. T., and FLORA, R. M., Induction
of tyrosine-a-ketoglutarate transaminase in rat
liver, dr. Biol. Chem., 1961, 236, 2699.
3. SCnlM'KE, R. T., SWEENEY, E. W., and BERLIN,
C. M., The roles of synthesis and degradation
in the control of rat liver tryptophan pyrrolase,
J. Biol. Chem., 1965, 240, 322.
4. SEGAL, H. L., and KIM, Y. S., Glucocorticoid
stimulation of biosynthesis of glntamic-
alanine transaminase, Proe. Nat. Avad. So.,
1963, 50, 912.
5. ROSEN, F., HARDING, H. R., MULttOLLAND, J.,
and NICHOL, C. A., Glucocorticoids and
transaminase activity, J. Biol. Chem., 1963,
238, 3725.
6. KNOX, W. E., OGATA, M., HASEGAWA,N., and
TOKUYAMA, K., The hormone and substrate
types of induction of liver tryptophan pyrrolase,
Sixth International Congress of Biochemistry,
1964, Washington, D.C., Secretariat Sixth
International Congress of Biochemistry, 9,
724.
7. VALENTINE, W. N., FOLLETTE, J. H., HARDIN,
E. B., BECK, W. S., and LAWRENCE, J. S.,
Studies on leukocyte alkaline phosphatase
activity : relation to stress and pituitary-adrenal
activity, d. Lab. Clin. Med., 1954., 44, 219.
8 TH~ JOURNAL OF CELL BIOLOGY • VOLIYME$9, 1966
effect on m e m b r a n e structure and the stability of
lysosomes (31). It is possible that the "sparing
effects" of prednisolone with respect to cell
damage induced by a number of environmental
agents, such as viruses, antigen-antibody reactions
(32), nutritional deprivation (33), and metabolic
inhibitors, are related to hormone-produced
changes in the cell membranes.
We wish to thank Mrs. Maude Levy for her superb
technical assistance.
This work was supported by Research Contract
U-1296 of the Health Research Council of the City
of New York and Genetics Training Grant 5 T1
HE 5307 of the United States Public Health Service.
During the course of the work, Dr. Griffin was a
Genetics Trainee.
Dr. Cox is a Career Scientist of the Health Re-
search Council of the City of New York.
Received for publication 19 July 1965.
8. CHmFFI, G., and CARFAGNA, M., The alkaline
phosphatase of intestinal epithelium of Bufo
vulgaris tadpoles during metamorphosis. The
influence of hydrocortisone on the epithelial
phosphatase in vitro, Acta Embryol. et Morphol.
Exp., 1960, 3, 213.
9. MOOG, F., The differentiation of enzymes in
relation to the functional activities of the
developing embryo, Ann. New York Acad. So.,
1952, 55, 57.
10. KATO, Y., The induction of phosphatases in
various organs of the chick embryo, Develop.
Biol., 1959, 1, 477.
11. Mooo, F., The adaptations of alkaline and acid
phosphatase in development, in Cells, Or-
ganisms and Milieu, (Dorothea Rudnick,
editor), New York, Roland Press Co., 1959,
121.
12. Cox, R. P., and MAcLEOD, C. M., Hormonal
induction of alkaline phosphatase in human
cells in tissue culture, Nature, 1961, 190, 85.
13. Cox, R. P., and MAGLEoD, C. M., Alkaline
phosphatase content and the effects of pred-
nisolone on mammalian cells in culture, Jr. Gen.
Physiol., 1962, 45, 439.
14. EAGLE, H., Amino acid metabolism in mam-
malian cell cultures, Science, 1959, 130, 432.
15. McLIMANS, W. F., DAVIS, E. V., GLOVER, F. L.,
and R~acE, G. W., The submerged culture of
mammalian cells: the spinner culture, or.
Immunol., 1957, 79, 428.
16. WAYMOUTH,C., Rapid proliferation of sublines of
 N.C.T.C. clone 929 (Strain L) mouse ceils in a
simple chemically defined medium (M.B.
752/1), J. Nat. Cancer Inst., 1959, 28, 1003.
17. Cox, R. P., and MAcLBoD, C. M., Repression of
alkaline phosphatase in h u m a n cell cultures by
cystine and cysteine, Proc. Nat. Acad. Sc., 1963,
49, 504.
18. LowRY, O. H., in Methods in Enzymology,
(S. P. Colowick and N. O. Kaplan, editors),
New York, Academic Press Inc., 1957, 4, 371.
19. LowRY, O. H., ROSEBOROUOH, N. J., FARR,
A. L., and RANDALL, R. J., Protein measure-
ment with the folin phenol reagent, J. Biol.
Chem., 1951, 193, 265.
20. BRAY, G. A., Liquid scintillator for aqueous
solution counting, Anal. Biochem., 1960, 1, 279.
21. KAPLOW, L., A histochemical procedure for
localizing and evaluating leucocyte alkaline
phosphatase activity in smears of bone marrow,
Blood, 1955, 10, 1023.
22. BURSTONE, M. S., Postcoupling, noncoupling and
fluorescence technique for the demonstration
of alkaline phosphatase, J. Nat. Cancer Inst.,
1960, 24, 1199.
23. Cox, R. P., and MAcLEoD, C. M., Regulation of
alkaline phosphatase in h u m a n cell cultures,
Cold Spring Harbor Syrup. Quant. Biol., 1964,
29, 233.
24. PARDEE, A. B., and PRESTIDOE, L. S., The
initial kinetics of enzyme induction, Bwchim. et
Biophysica Acta, 1961, 49, 77.
25. VARNER,J. E., and CHANDRA,G. R., Hormonal
control of enzyme synthesis in barley endo-
sperm, Proc. Nat. Acad. So., 1964, 52, 100.
M. J. GRIFFIN AND R. P. Cox
26. FRANKLIN,R. M., and BALTIMORE,D., Patterns
of macromolecular synthesis in normal and
virus-infected mammalian cells, Cold Spring
Harbor Syrup. Quant. Biol., 1962, 27, 175.
27. WEBBR, G., SINGHAL, R. L., STAMM, N. B., and
SRIVASTAVA, S. K., Hormonal induction and
suppression of liver enzyme biosynthesis,
Fed. Proc., 1965, 24, 745.
28. HONIG, G. R., and RABINOWITZ, N., Actinomycin
D: inhibition of protein synthesis unrelated to
effect on template R N A synthesis, Science,
1965, 149, 1504.
29. REVEL, M., and HIATT, H. H., The stability of
liver messenger RNA, Proc. Nat. Acad. Sc.
1964, 51, 810.
30. BLOOM, S., GOLDBERG, B., and GREEN, H., The
lifetime of messenger R N A for collagen and
cell protein synthesis in an established mam-
malian cell line, 1965, Biochem. and Biophys.
Research Comm., 19, 317.
31. WEISSMANN, G., and THOMAS, L., Studies on
lysosomes. II. The effect of cortisone on the
release of acid hydrolases from a large granular
fraction of rabbit liver induced by an excess of
vitamin A, J. Clin. Invest., 1963, 42, 661.
32. BUNIM, J. J., Corticotropin and corticosteroids,
in Chemistry, Physiology and Metabolic Effects
in Arthritis, (J. L. Hollander, editor) London,
Henry Kingston Publishers, 1960, 358.
33. ARPELS, C., BABCOCK,V. I., and SOUTHAM,C. M.,
Effect of steroids on h u m a n cell cultures;
sustaining effect of hydrocortisone, Proc. Soc.
Exp. Biol and Med., 1964, 115, 102.
Mechanism of Hormonal Induction. I 9