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

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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 by producing configurational changes in the
controlled in part by hormones. The mechanisms enzyme (1). Enzyme synthesis in cells also may be
of hormone action are probably complex and regulated by hormones. Recent evidence derived
include effects on cell permeability, alterations in from studies on several different enzyme systems
subcellular compartmentation, as well as changes such as tyrosine-a-ketoglutarate and glulamic-
in the activity of certain enzymes. It is likely that alanine transaminases and tryptophan pyrrolase
hormonal regulation of enzyme activity depends indicate that hydrocortisone promotes de novo
not only on the particular hormone but also on synthesis of these enzymes, while substrates
the enzyme system and its localization in the cell. promote an increase in the activity of these
Recent studies suggest several mechanisms enzymes by protecting them from cellular degrada-
whereby hormones may influence enzyme levels tion (2-6).
in cells. Alkaline phosphatase is induced by adrenal
Steroid hormones may influence the substrate glucocortcoids in human leukocytes in vivo (7)
specificity of certain enzymes (e.g., conversion of and, during embryonic development, in amphibian
glutamic dehydrogenase to alanine dehydrogenase) (8), chicken (9, 10) and mouse intestinal epi-
thelium (11). In certain h u m a n epithelial cell 2. Chemical Determinations
lines in tissue culture, the enzyme can also be
Cells were prepared for enzyme assay and protein
induced by hydrocortisone or its analogue pred- determinations as previously described (13, 17).
nisolone (12, 13). T h e s e dispersed cell cultures of Alkaline phosphatase activity of cell lysates was
either the monolayer or suspension type have measured spectrophotometrically with p-nitrophenyl
no a p p a r e n t intercellular orientation and are phosphate (8 mM) as substrate in 0.5 ~ 2-amino-2-
composed of a relatively homogeneous cell popu- methyl-l-propanol (AMP) buffer, pH 10.6. Specific
lation. Therefore, they have the advantage of being activity was expressed as #moles of p-nitrophenol
relatively simple cell systems for the study of hor- liberated in 15 rain per mg of protein (18). Acid
m o n e action and enzyme induction. phosphatase was determined by the method of Lowry
(18). Protein was assayed by Lowry's method (19).
T h e aims of the present study were: (a) to
determine the effect of prednisolone on the 3. Radioactive Isotope Incorporation
kinetics of synthesis of alkaline phosphatase in
Studies
suspension culture; (b) to examine the effects of
certain inhibitors of both R N A and protein In certain experiments, cultures were grown in
synthesis on induction of the enzyme in order to medium containing either leucine-l-Cn or uridine-
2-C 14. Two methods were used to measure radioac-
distinguish between its de novo synthesis and its
tive leucine or nridine which has been incorporated
protection from cellular degradation; (c) to
into cell protein and RNA, respectively.
estimate in a relatively simple cell system the MONOLAYER CULTURES : Cells grown in mono-
m i n i m u m stability of the messenger R N A for layer were harvested and the cell suspension was
alkaline phosphatase; and (d) to compare the washed twice in isotonic saline. The cells were then
effects of an adrenal glucocorticoid analogue on suspended at 4°C in 5% trichloroacetic acid (TCA).
cell multiplication in monolayer cultures and in The ceil residue was collected on Millipore HA filter
suspension cultures. and thoroughly washed with ice-cold 5% TCA.
The dried filters were placed on planchets and radio-
MATERIALS AND METHODS active measurements were made in a Nuclear Chi-
cago micromil window counter.
1. Cells and Media SUSPENSION CULTURES: Cells grown insuspen-
sion culture in medium containing radioactive iso-
Suspension cultures of HeLa S3 cells were grown topes were lysed with deoxycholate, and 0.2 ml of
in Eagle's minimal medium (MEM) without calcium lysate was mixed with 3.0 ml of ice-cold 1 N per-
ions (14). These cultures were furnished by Dr. chloric acid. The precipitate was sedimented at
Matthew Scharf of Albert Einstein College of Medi- 1,600 Rv~ in an International Refrigerated Centri-
cine. The methods of suspension culture were similar fuge at 0°C. The sediment was dissolved in 0.3 ml
to those described by McLimans et al. (15). For of 1 N NaOH, and 15 ml of dioxane scintillation
kinetic experiments, 10-ml aliquots of the cell su- solution were added (20). The sample was cooled
spension were removed at various times from the at 4°C in a polyethylene vial and then assayed for
spinner flasks. The volume of medium was main- radioactivity in a Packard Tricarb scintillation
tained by replacing the aliquots with fresh medium counter. The background radioactivity was 45 CPM
containing the same concentrations of compounds. and the counting efficiency was approximately 80%
This HeLa $3 cell line was also adapted to monolayer for C 14.
culture and was studied while growing in Eagle's
minimal essential medium (MEM) (14) and in 4. Cytology
Waymouth's medium (16), both containing 10%
Mitotic indices were determined on cells grown in
calf serum. Another HeLa S~ cell line, obtained from suspension culture. The cell suspension was washed
Dr. Harold Nitowsky of Johns Hopkins School of in isotonic saline and the cells were air dried on
Medicine, was grown in monolayer culture using covcrslips. Following fixation in 95% ethanol for
Waymouth's medium containing 10% human 10 to 15 rain, the coverslips were stained by the acetic-
serum. The methods of monolayer culture and the orcein method. The mitotic indices were determined
techniques of analysis used in this laboratory have by counting a minimum of 1500 nuclei per slide.
been described (13, 17). Experiments using mono- To determine alkaline phosphatase histochemi-
layer cultures were done in triplicate. All of the cell cally, cover slip preparations of HeLa $3 cells grown
lines used were free from pleuropneumonia-like in suspension culture were prepared as described
organism [PPLO] infection as determined by Dr. above. Alkaline phosphatase was determined histo-
Morton Davidson. chemically using Kaplow's modification of the Men-

2 TirE JOURNAL OF CELL BIOLOGY • VOLUME ~9, 1966


ten, Junge, Green method (21) or by the postcoupling during prednisolone induction and alter resus-
technique of Burstone (22). Both methods gave pending in medium without prednisolone was
similar results. determined semiquantitatively by histochemical
methods. Coverslip preparations were incubated
RESULTS with substrate until a faint staining of cells was
observed; the length of incubation and the intensity
Kinetics of Prednisolone Induction of Alkaline
of staining of individual cells correlate approxi-
Phosphatase in HeLa $3 Suspension Culture mately with their enzyme activity. Coverslip
Fig. 1 shows the kinetics of alkaline phosphatase preparations made from suspension cultures
induction by prednisolone in HeLa $3 suspension grown in medium containing prednisolone show
cultures. A representative experiment illustrates that the increase in enzyme activity is approxi-
three aspects of the induction curve as shown in mately the same throughout the cell population.
Fig. 1. During the first 18 hr of growth in medium On removing prednisolone from the medium, the
containing prednisolone, there is a small increase enzyme activity appears to decrease propor-
in the alkaline phosphatase activity up to twice tionately in all the cells. Acid phosphatase specific
the level observed in replicate cultures grown in activity remains unchanged during the experi-
medium without the hormone (controls). There- ment.
after, the rate of alkaline phosphatase induction Monolayer cultures of HeLa Sa cells in which
rises markedly and enzyme activity increases the enzyme is induced by prednisolone show
linearly, reaching a value about 10-fold greater results similar to those described above (13). The
than that of control cultures after 60 hr. The main difference between monolayer and sus-
rate of induction between 24 and 60 hr is 3.5 pension cultures is the magnitude of enzyme
times faster than that seen during the initial 24 hr. induction. During the first 15 hr, suspension
The plateau reached after 60 hr reflects the cultures show up to a 100% increase in alkaline
maximal enzyme activity induced by prednisolone phosphatase levels. However, most of the cell
under the conditions of this experiment. When lines studied in monolayer cultures show an
prednisolone-induced cultures are resuspended in increase in enzyme activity of only 10 to 20%
medium without prednisolone, enzyme levels during this time (13). The enzyme activity in-
decrease at a rate which is consistent with dilution duced by prednisolone in monolayer cultures is
by cell muhiplication. The relative alkaline 2- to 8-fold higher than control cultures, while
phosphatase activity of individual HeLa $3 cells 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 other hand, confluent 72-hr-old cultures con-
and Mitotic Index taining 4 to 5 million cells appeared cytologically
normal for the first 24 hr after adding puromycin,
In suspension cultures, prednisolone reduces and radioactive leucine incorporation was reduced
cell multiplication and increases the mitotic index by only 35% of the control value. Hormonal
as in the case of cells grown in monolayer cultures induction of alkaline phosphatase was markedly
(13). There was no apparent change, during the reduced, however.
first 48 hr, in the generation time of 25 4- 5 hr in Cultures grown in medium containing both
suspension cultures grown in medium containing puromycin and prednisolone showed less morpho-
prednisolone. However, at about 60 hr, the logical damage as compared to replicate cultures
generation time is increased to 45 4- 5 hr and the grown in medium with puromycin alone. The
mitotic index of 8 to 9 % is twice that of replicate steroid hormone appears to protect cells from the
cultures grown in the absence of hormone. When cytotoxic effects of puromycin without interfering
prcdnisolone-induced cultures are resuspcnded in with the inhibition of protein synthesis by the
m e d i u m without prcdnisolone within 8 to 16 hr, drug.
the generation time returns to 25 4- 5 hr. Table II shows the effects of two concentrations
Effects of Puromycin and Actinomycin D on (0.025 #g per ml and 0.10 t~g per ml) of Actino-
mycin D on prednisolone induction of alkaline
Prednisolone Induction of Alkaline phosphatase in H e L a $8 monolayer cultures.
Phosphatase The Actinomycin D at the lower concentration
HELA S3 MONOLAYER CULTURES GROWN IN did not affect alkaline phosphatase induction,
WAYMOUTH'S M E D I U M : For the first 12 to even when added at the same time as prednisolone.
18 hr of growth in medium with prednisolone, This concentration of Actinomycin D reduced the
there is little or no increase in alkaline phosphatase incorporation of phosphate-P s2 into cellular
(13). During the following 24 to 36 hr, the enzyme macromolecules such as nucleic acids by 35%.
levels increase rapidly. Therefore, in the experi- Increasing the Actinomycin D concentration to
ments described in Table I, puromycin was added 0.10 ~g per ml Mocked hormonal induction of
18 to 20 hr after adding prednisolone so that the alkaline phosphatase and reduced phosphate-P 32
effects of the inhibitor could be observed during incorporation by 68 %.
the phase of rapid enzyme increase. The cultures Table I I I shows that Actinomycin D at the
were harvested 24 hr after the addition of puro- concentration which blocks induction when added
mycin. at the same time as prednisolone is without effect
Table I shows that puromycin at 0.1 #g per ml when added 18 hr after the hormone.
did not affect either alkaline phosphatase induction HELA S3 CELL SUSPENSION CULTURES:
or leucine-C 14 incorporation. At a higher concen- Fig. 2 shows the effect of adding either puromycin
tration of puromycin, 0.5 #g per ml, the pred- at 5.0 #g per ml or Actinomycin D at 5.0 #g per
nisolone-induced increase in alkaline phosphatase ml to suspension cultures during the phase of
and the incorporation of leucine-C 14 into protein rapid linear synthesis of the enzyme. When puro-
were markedly reduced. The effect of puromycin mycin, an inhibitor of protein synthesis, is added
on alkaline phosphatase induction depends not to the cultures, prednisolone induction of alkaline
only on the concentration of the inhibitor but also phosphatase promptly ceases and the specific
on the confluency of the monolayer. As shown in enzyme activity falls. The experimental results
Table I, recently transferred cultures containing have been plotted for only the first 12 hr after
about 1 million cells when grown in the presence adding puromycin. This concentration of puro-
of puromycin at 0.5 #g per ml showed evidence of mycin irreversibly damages cells within 7 to 10
cell injury within 12 to 14 hr. In these cultures, hr. After 12 hr, the cells show altered microscopic
the alkaline phosphatase was decreased by one- appearance with increased cytoplasmic granularity
half while the acid phosphatase was markedly and ballooning, as well as the presence of cell
reduced. Acid phosphatase, a constitutive enzyme fragments and debris. These cells have lost the
in cell cultures, is a sensitive indicator of cell ability to exclude a 0.15 % solution of trypan blue.
damage. Radioactive leucine incorporation was Table IV shows the effect of puromycin at 5.0
reduced by 70 to 9 0 % in these cultures. O n the #g per ml on the leucine-C I4 incorporation into

4 THE JOURNALOF CELL BIOLOGY • VOLUME ~9, 1966


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 39 hr after prednisolone reduced uridine-C 14 in-
puromycin, and further induction of alkaline c o r p o r a t i o n into nucleic acids by 9 5 % (Table
phosphatase was completely blocked. IV). However, the rate of alkaline phosphatase
Fig. 2 also shows the effects of Actinomycin D synthesis and the increase in total cell protein were
at 5.0 /~g per ml on prednisolone induction of 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 that de novo protein synthesis is involved in the
of irreversible cell damage. induction of alkaline phosphatase by prednisolone.
T h e effects of a low concentration of Actino- Puromycin, an inhibitor of the final steps of
mycin D (0,05 /zg per ml) were also studied at 8 protein synthesis, immediately blocks further
hr and at 39 hr after adding prednisolone to prednisolone induction of enzyme when added to
suspension cultures of H e L a $3 cells. This concen- cultures during the rapid phase of enzyme increase.
tration after 12 hr reduced radioactive uridine In contrast, Actinomycin D (5.0/zg per ml) added
incorporation into the nucleic acids of H e L a during the rapid phase of enzyme induction does
cell suspension cultures by only 10 to 50%. not interfere with enzyme synthesis for at least 12
Alkaline phosphatase induction and increase in hr following its addition to suspension cultures.
total cell protein were not affected for at least This concentration of inhibitor is known to act
12 hr. However, morphological evidence of injury within an hour (26) and reduce by 9 5 % the
similar to that observed with the high concen- incorporation of radioactive label into R N A .
tration of actinomycin (5.0 #g per ml) was seen This result is expected from studies on other
within 12 to 16 hr after adding the inhibitor. biological systems which have shown that Actino-
mycin D markedly inhibits DNA-dependent
DISCUSSION R N A synthesis and to a lesser degree the synthesis
T h e kinetics of prcdnisolone induction of alkaline of protein (27, 28). Thus, previously formed
phosphatase in human cell cultures differs somc- messenger R N A will be insensitive to the effects
what from the kinetics of enzyme formation in of Actinomycin D and will be capable of directing
bacteria after adding inducer. For example, during further protein synthesis until it is degraded. The
~-galactosidase induction in E. coli, enzyme syn- stability of this messenger R N A can be approxi-
thesis begins within 3 min after adding inducer, mated as at least 10 to 12 hr, since Actinomycin D
and the rate of increase of enzyme is lincar (24). at a concentration that completely inhibits new
O n the other hand, in H e L a cells there appear to R N A synthesis does not interfere with induction
be two different rates of alkaline phosphatase for this period of time. The lifetime of certain other
synthesis which occur in sequence: an initial slow R N A messengers in m a m m a l i a n cells is known to
rate of increase of enzyme is followed from 15 to be long (29, 30).
24 hr later by an abrupt, rapid linear synthesis. An intriguing aspect of the current studies is
Similar biphasic enzyme induction after addition the effect of prednisolone on the generation time
of hormone has also been observed in plant seeds and mitotic index of H e L a Ss cell cultures. These
(25). The rapid phase of gibbcrcllic acid induction cells exhibit an increased generation time with
of a-amylase in barley endosperm is preceded by increased cytoplasmic growth and doubling of the
a 9- to 15-hr slow phase. mitotic index. There appear to be a dispropor-
The data presented in this study strongly suggest 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 effect on m e m b r a n e structure and the stability of
hours of resuspending the cells in medium without lysosomes (31). It is possible that the "sparing
prednisolone. Semiquantitative estimation of effects" of prednisolone with respect to cell
alkaline phosphatase activity by histochemical damage induced by a number of environmental
methods shows that ceils in mitosis appeared to agents, such as viruses, antigen-antibody reactions
stain slightly more intensely than cells in inter- (32), nutritional deprivation (33), and metabolic
phase. However, all cells from cultures grown in inhibitors, are related to hormone-produced
medium with prednisolone showed a marked changes in the cell membranes.
increase in alkaline phosphatase staining when
compared to cells grown in medium without the We wish to thank Mrs. Maude Levy for her superb
hormone. technical assistance.
Prednisolone protects cells from the cytotoxic This work was supported by Research Contract
effects of both puromycin and Actinomycin D U-1296 of the Health Research Council of the City
without affecting the inhibition of protein and of New York and Genetics Training Grant 5 T1
R N A syntheses. The mitigation by prednisolone HE 5307 of the United States Public Health Service.
of this cellular injury may be similar to the 24-hr During the course of the work, Dr. Griffin was a
delay in the cytopathic effects of poliovirus in- Genetics Trainee.
fection observed in H e L a cell cultures grown in Dr. Cox is a Career Scientist of the Health Re-
medium with prednisolone (13). Adrenal gluco- search Council of the City of New York.
corticoids are also known to have a profound Received for publication 19 July 1965.

REFERENCES

1. TOMKIN, G. M., and YEILDING, K. L., Regula- 8. CHmFFI, G., and CARFAGNA, M., The alkaline
tion of the enzymic activity of glutamic dehy- phosphatase of intestinal epithelium of Bufo
drogenase mediated by changes in its structure, vulgaris tadpoles during metamorphosis. The
Cold Spring Harbor Syrup. Quant. Biol., 1961, influence of hydrocortisone on the epithelial
26, 331. phosphatase in vitro, Acta Embryol. et Morphol.
2. KEN•EY, F. T., and FLORA, R. M., Induction Exp., 1960, 3, 213.
of tyrosine-a-ketoglutarate transaminase in rat 9. MOOG, F., The differentiation of enzymes in
liver, dr. Biol. Chem., 1961, 236, 2699. relation to the functional activities of the
3. SCnlM'KE, R. T., SWEENEY, E. W., and BERLIN, developing embryo, Ann. New York Acad. So.,
C. M., The roles of synthesis and degradation 1952, 55, 57.
in the control of rat liver tryptophan pyrrolase, 10. KATO, Y., The induction of phosphatases in
J. Biol. Chem., 1965, 240, 322. various organs of the chick embryo, Develop.
4. SEGAL, H. L., and KIM, Y. S., Glucocorticoid Biol., 1959, 1, 477.
stimulation of biosynthesis of glntamic- 11. Mooo, F., The adaptations of alkaline and acid
alanine transaminase, Proe. Nat. Avad. So., phosphatase in development, in Cells, Or-
1963, 50, 912. ganisms and Milieu, (Dorothea Rudnick,
5. ROSEN, F., HARDING, H. R., MULttOLLAND, J., editor), New York, Roland Press Co., 1959,
and NICHOL, C. A., Glucocorticoids and 121.
transaminase activity, J. Biol. Chem., 1963, 12. Cox, R. P., and MAcLEOD, C. M., Hormonal
238, 3725. induction of alkaline phosphatase in human
6. KNOX, W. E., OGATA, M., HASEGAWA,N., and cells in tissue culture, Nature, 1961, 190, 85.
TOKUYAMA, K., The hormone and substrate 13. Cox, R. P., and MAGLEoD, C. M., Alkaline
types of induction of liver tryptophan pyrrolase, phosphatase content and the effects of pred-
Sixth International Congress of Biochemistry, nisolone on mammalian cells in culture, Jr. Gen.
1964, Washington, D.C., Secretariat Sixth Physiol., 1962, 45, 439.
International Congress of Biochemistry, 9, 14. EAGLE, H., Amino acid metabolism in mam-
724. malian cell cultures, Science, 1959, 130, 432.
7. VALENTINE, W. N., FOLLETTE, J. H., HARDIN, 15. McLIMANS, W. F., DAVIS, E. V., GLOVER, F. L.,
E. B., BECK, W. S., and LAWRENCE, J. S., and R~acE, G. W., The submerged culture of
Studies on leukocyte alkaline phosphatase mammalian cells: the spinner culture, or.
activity : relation to stress and pituitary-adrenal Immunol., 1957, 79, 428.
activity, d. Lab. Clin. Med., 1954., 44, 219. 16. WAYMOUTH,C., Rapid proliferation of sublines of

8 TH~ JOURNAL OF CELL BIOLOGY • VOLIYME$9, 1966


N.C.T.C. clone 929 (Strain L) mouse ceils in a 26. FRANKLIN,R. M., and BALTIMORE,D., Patterns
simple chemically defined medium (M.B. of macromolecular synthesis in normal and
752/1), J. Nat. Cancer Inst., 1959, 28, 1003. virus-infected mammalian cells, Cold Spring
17. Cox, R. P., and MAcLBoD, C. M., Repression of Harbor Syrup. Quant. Biol., 1962, 27, 175.
alkaline phosphatase in h u m a n cell cultures by 27. WEBBR, G., SINGHAL, R. L., STAMM, N. B., and
cystine and cysteine, Proc. Nat. Acad. Sc., 1963, SRIVASTAVA, S. K., Hormonal induction and
49, 504. suppression of liver enzyme biosynthesis,
18. LowRY, O. H., in Methods in Enzymology, Fed. Proc., 1965, 24, 745.
(S. P. Colowick and N. O. Kaplan, editors), 28. HONIG, G. R., and RABINOWITZ, N., Actinomycin
New York, Academic Press Inc., 1957, 4, 371. D: inhibition of protein synthesis unrelated to
19. LowRY, O. H., ROSEBOROUOH, N. J., FARR, effect on template R N A synthesis, Science,
A. L., and RANDALL, R. J., Protein measure- 1965, 149, 1504.
ment with the folin phenol reagent, J. Biol. 29. REVEL, M., and HIATT, H. H., The stability of
Chem., 1951, 193, 265. liver messenger RNA, Proc. Nat. Acad. Sc.
20. BRAY, G. A., Liquid scintillator for aqueous 1964, 51, 810.
solution counting, Anal. Biochem., 1960, 1, 279. 30. BLOOM, S., GOLDBERG, B., and GREEN, H., The
21. KAPLOW, L., A histochemical procedure for lifetime of messenger R N A for collagen and
localizing and evaluating leucocyte alkaline cell protein synthesis in an established mam-
phosphatase activity in smears of bone marrow, malian cell line, 1965, Biochem. and Biophys.
Blood, 1955, 10, 1023. Research Comm., 19, 317.
22. BURSTONE, M. S., Postcoupling, noncoupling and 31. WEISSMANN, G., and THOMAS, L., Studies on
fluorescence technique for the demonstration
lysosomes. II. The effect of cortisone on the
of alkaline phosphatase, J. Nat. Cancer Inst.,
release of acid hydrolases from a large granular
1960, 24, 1199.
fraction of rabbit liver induced by an excess of
23. Cox, R. P., and MAcLEoD, C. M., Regulation of
alkaline phosphatase in h u m a n cell cultures, vitamin A, J. Clin. Invest., 1963, 42, 661.
Cold Spring Harbor Syrup. Quant. Biol., 1964, 32. BUNIM, J. J., Corticotropin and corticosteroids,
29, 233. in Chemistry, Physiology and Metabolic Effects
24. PARDEE, A. B., and PRESTIDOE, L. S., The in Arthritis, (J. L. Hollander, editor) London,
initial kinetics of enzyme induction, Bwchim. et Henry Kingston Publishers, 1960, 358.
Biophysica Acta, 1961, 49, 77. 33. ARPELS, C., BABCOCK,V. I., and SOUTHAM,C. M.,
25. VARNER,J. E., and CHANDRA,G. R., Hormonal Effect of steroids on h u m a n cell cultures;
control of enzyme synthesis in barley endo- sustaining effect of hydrocortisone, Proc. Soc.
sperm, Proc. Nat. Acad. So., 1964, 52, 100. Exp. Biol and Med., 1964, 115, 102.

M. J. GRIFFIN AND R. P. Cox Mechanism of Hormonal Induction. I 9