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Estrogen synthetase stimulation by hemin in human choriocarcinoma cell culture
Vol. 127, No. 1, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 232-238
February 28, 1985
ESTROGEN
SYNTHETASE
S T I M U L A T I O N BY HEMIN CELL CULTURE
Francis 1Dept. 2Dept.
of
L.
Biol.
Gyn/Obs.,
Received December
i0,
Bellino Sci.,
Med.
I
and
Robert
SUNY/Buffalo,
Co11.
of
HUMAN CHORIOCARCINOMA
IN
O.
Hnssa 2
Buffalo,
Wisconsin,
NY 1 4 2 6 0
Milwaukee,
WI 5 3 2 2 6
1984
Summary: The ability of hemin to stimulate estrogen synthetase (aromatase) in cultured human trophoblast cells and in cellular homogenates was investigated and compared with aromatase stimulation by dibutyryl cAMP [ ( B u ) 2 c A M P ] . Cells grown with hemin for 24 h, or homogenates incubated for 45 min with hemin, showed maximal aromatase stimulation ( 1 5 0 t o 200% o f a c t i v i t i e s in the absence of hemin) at 25 ~M a n d 0 . 1 ~M, r e s p e c t i v e l y . Aromatase stimulation in culture by 25 ~M h e m l n w a s o b s e r v e d within 4 h after hemin addition, while (Bu)2cAMP required more than 6 h. Intracellular heme and porphyrin levels were higher (160 to 185%) in 96 h (Bu)2oAMP-grown cells than control cells. ® 1985 Acad~ic Press, Inc.
Regulation estrogen
as
the
synthetase
physiological and
of
is
of
processes, clinical
P-450
of
the
enzyme
mechanism In
one
(Bu)2cAMP
plays as
in
pharmacological
its
plus
(3,4),
system,
protein
(5).
component
several
androgens,
important
role
in
and
fetal
pathological There
are
inhibition
(1,2)
relatively
few
many
development,
processes,
many
basic
of
this
studies
choriocaroinoma
h by
aromatase
Abbreviations used: (Bu)2cAMP iB~u)~ p-~uus T mM t h e o p h y l l i n e .
0006-291X/85 $1.50 Copyright © 1985 ~ A c a ~ m ~ Press, ~c. AH righ~ ~ repro~c~n m aM ~ r m rese~eK
aromatase
a process
Cytochrome of
from
reproduction
stimulate
96
an
estrogens
and
such clinical
cytochrome of
the
in
culture,
regulation.
human
theophylline over
synthesis
but
physiological
model
makes
carcinomas.
eight-to-ten-fold
regulatory
such
interest
system
of
which
(aromatase),
estrogen-dependent
studies
enzyme
P-450 in
these
dibutyryl
232
cells activity
requiring
RNA a n d
appears
to
cells
(5,6).
cAMP,
dbT
be
-
the We r e p o r t
I
mM
VOI. 127, No. 1, 1985
here
the
aromatase of
hemin
cells
in
ability in to
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of
these
to
cells.
stimulate
culture
hemin
as
a well
rapidly This
and
is
the
steroidogenic as
in
first cytochrome
cellular
KATERIALS
significantly report P-450
stimulate of
the enzyme
ability in
homogenates.
AND METHODS
~ ! $ ! ~ : The arom~tase subst~]te, [l~-3H, 4-14C] androstenedione (64.4 Ci -H, 1.7 Ci --C per mol), was prepared using the drastic alkali treatment (7) on a m~ture of [I,2"~H] androstenedione (41 Ci/mmol f r o m NEN) a n d [ 4 ~ ' C ] androstenedione (57.4 Ci/mol from NEN). The distribution of H is 77~ in the i~ position (by nmr from NEN). Hemin (Type I, bovine), protoporphyrin IX, oxalic acid, non-radioactive steroids, cytochrome c a n d NADPH w e r e p u r c h a s e d from Sigma Chemical Co. Liquiscint scintillation cocktail came from National Diagnostics. GIBCO s u p p l i e d the cell culture media and serum. Methods: Cell Culture: Human choriocarcinoma cells (JAr line) were maintained in continuous culture with growth medium changed daily, and grown in the absence or presence of dbT as previously described (8). The appropriate amount of hemin (dissolved under reduced light conditions i n 0 . 1 M NaOH, b r o u g h t to approximate pH 8 . 5 w i t h 0.1 N HC1 a n d f u r t h e r diluted with culture medium to the proper concentration) was maintained in the cell culture medium in the dark for the time periods indicated. At termination, the attached cells were washed twice using serum-free culture medium containing the appropriate amount of hemin, removed from the flask and collected by centrifugation as described (9). Enzyme and Protein Ass~x~: Frozen cell pellets were thawed and homogenized i n GP b u f f e r (5). NADPH-cytochrome c reductase activity (5) and protein content (10) were measured as described. The aromatase assay,~performed in duplicate o~ t r i p l i c a t e , was based on the transfer of "H from the substrate t o ~H_O 5 ~oncomitant with estrogen production (4), using subs~rate ( 1 0 dpm "H, 200 ng androstenedione), 0 . 5 mg NADPH a n d h o m o g e n i z e d cells (0.8 t o 2 mg p r o t e i n ) in a total volume of 1 ml. Control experiments verified substrate3saturation and linearity of the assay. The assay was conducted and H20 quantitated as described (9). We v e r i f i e d in selected samples that the apparent activation of aromatase by hemin using the -H20 assay is correlated with an increase in radiolabeled estrogen production (procedure as in (11)). ~ ~ h X ~ ! ~ _ ~ ! ! X : Cellular heme and porphyrin content were measured by the method of Granick et a!.(12) and Sassa et !~. (13), modified as described here, after washing the cells twice in 0.15 M NaC1. The cell pellet was homogenized in 0.067 M phosphate buffer, pH 7 . 4 , and aliquots mixed with 1 ml o x a l i c acid (saturated solution at 21°C) for heme measurement or extracted with 1 ml o f PM [ m i x t u r e of 1 N perchloric acid-methanol (1:1, v/v)] for porphyrin measurement. For the heme assay, samples were heated at 100°C for 30 min, 0 . 5 ml o f PM a d d e d after cooling to room temperature, and the 1,000 xg-20 min supernatant assayed. Fluorescence was measured in an Aminco-Bowman spectrofluorometer, with a red-insensitive photomultiplier tube (1P21, $4 s p e c t r a l response, 0 . 2 mm s l i t ) , a t 4 0 6 nm excitation (slit-22nm) and emissions (slit-11 nm) o f 5 7 7 - 5 7 8 nm (minimum) a n d 5 9 6 nm ( m a x i m u m ) . Control experiments showed that the 233
Vol. 127, No. 1, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
fluorescence was linearly related to the amount of protein in the assay over the range utilized. Statistical Analxsis: All experiments described here were performed at least twice. Except where otherwise noted, all assays were in duplicate or triplicate and the results reported as mean±S.D. Statistical comparisons were made using Student's t-test.
RESULTS AND D I S C U S S I O N Various control
concentrations
and
dbT-grown
activities
of
determined
(Fig.
specific to
and
about failed
10
aromatase 1).
in
the
~M f o r
to
and The
were cells
hemin
stimulation
absence
incubated in
NADPH-cytochrome
optimal
dbT-srown
affect
hemin
choriocarcinoma
significant
aromatase
of
of cells.
was
This
25
optimal
secretion
of
human
glycoprotein
hormone
u-subunit
or
progesterone
cells
unpublished
virus-transformed concentrations
18ot-
erythroleukemia above
IA
work).
.
.
!
100
.
~M w a s
.
As
.
in to
for
relative cells
concentration
control
mouse
culture
these
0.00S
gonadotropin, from
reported
the
control
hemtn
chorionic
cells toxic
(p(
specific
were for
~M f o r
h with
and
reductase
aromatase
the
(R.H.,
culture
concentration
of
hemin)
c
24
for
(14),
cells.
Friend hemin These
data
B
/
"o ~ 140i ._~ o 100~-
-
,5 10
50 100
,500 1000 5 10 Hernin Concentrotion (p.M)
-
50
100
Fig. I. Aromatase stimulation by h c m i n i n c u l t u r e d cells. (A) Choriocarcinoma c e l l s w e r e g r o w n w i t h medium c o n t a i n i n g hemin as indicated. (B) Same a s (A) e x c e p t dbT was i n c l u d e d in the medium. The I 0 0 ~ ( h e m i n - f r e e control) v a l u e s f o r a r o m a t a s e (X; p m o l / m l n / m g protein), NADPH-cytoehrome c reductasc (• ; n m o l / m l n / m g protein), and protein (e;mg/flask) w e r e f o r (A) 0 . 2 3 , 2 . 5 8 a n d 5 . 5 , r e s p e c t i v e l y , and f o r (B) 0 . 9 5 , 2 . 5 5 a n d 5 . 5 , r e s p e c t i v e l y . These data are a c c u m u l a t e d f r o m and r e p r e s e n t a t i v e of five separate determination for control cells and two f o r d b T - g r o w n c e l l s . 234
and
Vol. 127, No. 1, 1985
show
for
hemin
the
of
first
mechanism of
following
the
1 do
treatment P-450
performed
in
mechanism
of
insertion
into
mechanism
of
trations that
action.
with
agents
(15)
or
that
in
cells
extent
of
aromatase
cultured
cells
occurs
at
0.1
together
suggest
values
in
aromatase
the
absence
cytochrome
by
a
Both
hemin)
P-450
in
results
these
that
sets
of
aromatase
by
activation
suggests
that
cells
is
to
hemin
as
about in
the
(16),
fractions,
We t e s t e d
activation
of
of
regarded
The
tissues
tissues
various
X ~ -
activates
of
hepatic
formation
(17,18).
the
stimulation
subcellular
incubating
extent
the
in
generally
~M h e m i n .
the
of
the
and/or
~n
similar
Furthermore,
of
of
non-hepatic
P-450
a
mechanism.
studies
promote
is
by
hemin
and
identification
untreated
activation
activation
that
stimulation
cells
hydzoxylation,
homogenates
homogenized
knowledge,
cultured
previous
apocytochzome
hemin
our
permit
drug
with
hemin
the
in
not
Since
tissue
of
enzyme.
P-450-dependent
apocytochrome were
in
Fig.
best
enzyme
P-450
heme
cytochzome
P-450
the
cytochrome
results of
to
time,
a cytochrome
steroidogenic The
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in seen data
an
be
X~!
this concenFig.
2
with taken
insertion
(150
to
200%
of
30
to
50% o f
apocytochzome
form.
o
200
§ "5 150
g ~
100,
~
50
o
o o
,
I
200
,
I
400
,
I
600
,
I
800
,
I
1000
[Heroin] (nM) Fig. 2. Aromatase stimulation by hemin in cellular homogenates. Control choriocarcinoma cells were homogenized and incubated with hemin as indicated a n d 1 . 5 mM EDTA f o r 45 m i n a t 3 7 ° C i n f l a s k s protected from light. The aromatase essay was initiated by adding substrate a n d NADPH f o l l o w e d by incubation a t 3 7 ° C f o r 32 m i n . The control (100%) aromatase activity was 0.055 pmol/min/mg protein.
235
show
the
Vol. 127, No. 1, 1 9 8 5
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
350
/
= 300 ~6 250 200 ~150 o o 100 '< 5o
O0
I
I
2
i
I
4
,
I
/
J
6 Time (hr)
"
2
~4
Fig. 3. Time c o u r s e of h e m i n - and d b T - s t i m u l a t i o n of aromatase in culture. Cells were grown in medium alone (control), medium with 25 ~M h e m i n ( . ) , o r m e d i u m w i t h dbT ( [] ) . Aromatase specific activities i n h e m i n - a n d dbT - c o n t a i n i n g medium were divided by the specific activity of control cells at each time point. The a v e r a g e NADPH-cytochrome c reductase activity or protein content throughout t h e 24 h w e r e n o t s i g n i f i c a n t l y different in control or treated cells. The c o n t r o l aromatase activities (pmol/min/mg protein) at zero time averaged 0.25-+ 0.012 (n=10), increasing to 0.46 + 0.006 (n=4) at 6 h before declining to 0.3+0.005 ( n = 4 ) a t 24 h .
To
determine
stimulation
of
aromatase (Fig. but
3). not
48
the
aromatase
At in
h.
4
and
control
By 2 4 in
h
cells
grown
dbT.
The when
higher relative
in
aromatase
levels to
of control
25
FM h e m i n
aromatase 24
sought
and
to
activity and
dbT
activity
in
significantly
in
cells
hemin-grown than
cells, the
value
cells
from
24
exceeded
cells. if
by
intracellular
of
time
without
that
only
comparing
stimulated observed
236
of
cultured
dbT-growu
dbT
cells.
in
course
a maximum
to
porphyrin
time
higher
reaching
determine
suggest is
well-documented the
pM h e m i n - g r o w n
periods I
the
by
activity
responsive
Table
in
compared
(p<0.00$),
and
heme
we
was
various
data
dbT,
cells,
are
role
by
aromatase
we
for
a
6 h,
the
control
levels
plays
cells
Furthermore, porphyrin
by
dbT-grown
in
value
dbT,
heme
stimulation
activity to
if
by
heme
these
levels
(control)
after 8-to in
96
h
or
in
with
growth
10-fold
dbT-grown
and
(5), cells
with were
Vol. 1 2 7 , No. 1 , 1 9 8 5
Table
1.
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
Porphyrin
a n d heme
content
of
choriocarcinoma
Porphyrin time
control
cells
Heme
dbT-grown
control
dbT-grown
pmol/mg
pmol/mg prot.
prot.
8.15 ± 2.28
13.45+_ 1 . 2 3 *
Experiment
I
96 h
0.51
+_0.15
0.94 ± 0.42
Experiment
II
48
h
1.55
+_0.31
1 . 2 5 -+ 0 . 0 9 8
11.6
+_ 0 . 7 1
13.3_+ 0 . 4 7 *
72 h
0.92
±0.07
1.2
11.3
+_ 1 . 3 5
11.9+_ 0 . 9 6
96
0.72
+_0.1
1.34 + 0.11"
h
± 0.063*
8 . 4 3 _+ 0 . 5 4
1 3 . 3 _+ 0 . 7 7 *
C e l l s w e r e g r o w n i n m e d i u m a l o n e o r w i t h dbT f o r t h e i n d i c a t e d time, end then washed and collected as d e s c r i b e d . Aliquots of homogenized cells were a s s a y e d f o r heme a n d p o r p h y r i n content end protein concentration. Porphyrin was undetectable in both growth medium and undiluted serum. Eeme l e v e l s w a s 1 . 5 7 pM i n 10% s e r u m . The t o t a l heme c o n t e n t of the growth medium per flask divided by the total cellular protein attached to the flask i s a b o u t 2 . 4 nmol heme/mg p r o t e i n . • v (
0.005
relative
Provided 10
to
20%
of
consistent involve the to
i~
that the
heme
intracellular
~K~
the
regulation
of
the that
apocytochrome significant culture
enzyme.
of
is
aromatase and
i~
heme
in
vitro.
Since
1
these
this is
data
dbT
less
than
are by
heme
dbT
supply
apocytochrome
unique
limiting
by
are
stimulation
aromatase
assumptions
stimulation
levels
intracellular
into
--
I
aromatase
heme
present
time.
P-450
the
Implicit
intracellular
P-450
same
levels that
insertion
at
eytochrome
hypothesis
assumptions
in
aromatase
cells
the
activate
hemin
control
with
subsequent
and
to
that
supported
optlmal
are
the
aromatase by
the
concentrations
stimulates
and P-450
hypothesis and
may
aromatase
rapid of to
a
Direct estimates of the total cytochrome P-450 in homogenized, subcellular-fraetionated JAr cells w e r e 3 a n d 13 p m o l / m g protein in the low-speed pellet from control and dbT-grown cells, respectively, with undetectable levels ((2 pmol/mg protein) in the eel1 homogenate and microsomal fractions (8). Aromatase cytochrome P-450 constitutes only a fraction of the total cytochrome P-450. By extrapolating cytochrome P-450 levels from human placental microsomes to JAr cells based on the relative aromatase specific activity, we estimate aromatase cytochrome P-450 levels in JAr cells at about 0.1 and 1 pmol/m 8 protein in control and dbT-grown cells, respectively. 237
Vol. 127, No. 1, 1985
much g r e a t e r in
the
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
extent
mechanism
of
than
hemin,
additional
factors
must
be
involved
dbT a c t i o n .
Acknowledgments: We t h a n k Mr. 7 . K u r t z , Ms. A. R u c k e r t , M r s . M. R i n k e a n d Ms. M.McAsey f o r t e c h n i c a l assistance and Mrs. U.Brunn a n d Mr. 7 . S t a m o s f o r a s s i s t a n c e with the manuscript. Supported by U.S.P.H.S.Grant HD 1 6 5 9 3 .
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16. 17.
Flynn, G.A., 7ohnston, 7.0., Wright, C.L., and Metcalf, B.W. ( 1 9 8 1 ) B i o c h e m . B i o p h y s . R e s . Commun. 1 0 3 , 9 1 3 - 9 1 8 . Harris, A.L., Powles, T.7., and Smith, I.E. (1982) Cancer Res. 42, 3405s-3408s. Zachariah, P . K . , a n d 7 u c h a u , M.R. ( 1 9 7 7 ) 7 . S t e r o i d B i o c h e m . 8, 221-228. Bellino, F . L . ( 1 9 8 2 ) . 7. S t e r o i d Biochem. 17, 261-270. Bellino, F.L., a n d H u s s a , R.O. ( 1 9 8 1 ) S t e r o i d s 37, 51-61. Bellino, F.L., and H u s s a , R.O. (1978) Biochem. B i o p h y s . Res. Commun. 8 5 , 1 5 8 8 - 1 5 9 5 . O s a w a , Y . , a n d S p a e t h , D. ( 1 9 7 1 ) B i o c h e m i s t r y 10, 66-71. Bellino, F.L., and H u s s a , R.O. (1982) E n d o c r i n o l o g y 111, 1038-1044. Bellino, F.L., H u s s a , R . O . , a n d O s a w a , Y. ( 1 9 7 8 ) S t e r o i d s 32, 37-44. Bellino, F.L., Gilani, S.S.H., Eng, S.S., Osawa, Y., and Duax, W.L. ( 1 9 7 6 ) B i o c h e m i s t r y 15, 4730-4736. Lobo, 7.0., Bellino, F.L., and Bankert, L. ( 1 9 8 5 ) E n d o c r i n o l o g y , in press. Granick, S., Sinclair, P . , S a s s a , S. a n d G r i e n i n g e r , G. ( 1 9 7 5 ) 7 . B i o l . Chem. 250, 9215-9225. Sassa, S., Granick, S . , C h a n g , C . , a n d K a p p a s , A. ( 1 9 7 4 ) i n Erythropoiesis (Nakao, K., Fisher, 3., and Takaku, F., eds.) p.383, Univ. Park Press. Chang, C,.S., a n d S a s s a , S. ( 1 9 8 2 ) 7 . B i o l . Chem. 2 5 7 , 3 6 5 0 - 3 6 5 4 . Correia, M . A . , a n d M e y e r , U.A. ( 1 9 7 5 ) P r o c . N a t . A c a d . S c i . USA 72, 400-404. Omiecinski, C.7., Chao, S.T., a n d 7 u c h a u , M.R. ( 1 9 8 0 ) D e v . Pharmacol. T h e r . 1, 9 0 - 1 0 0 . Correia, M.A., Farrell, G . C . , S c h m i d , R . , O r t i z de M o n t e l l a n o , P . R . , Y o s t , G . S . , and Mico, B.A. ( 1 9 7 9 ) 7. B i o l . Chem. 2 5 4 , 15-17.
18.
W a g n e r , G . C . , P e r e z , M., T o s c a n o , W . A . , ( 1 9 8 1 ) 7. B i o l . Chem. 2 5 6 , 6 2 6 2 - 6 2 6 5 .
238
7r.,
and Gunsalus,
I.C.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 232-238
February 28, 1985
ESTROGEN
SYNTHETASE
S T I M U L A T I O N BY HEMIN CELL CULTURE
Francis 1Dept. 2Dept.
of
L.
Biol.
Gyn/Obs.,
Received December
i0,
Bellino Sci.,
Med.
I
and
Robert
SUNY/Buffalo,
Co11.
of
HUMAN CHORIOCARCINOMA
IN
O.
Hnssa 2
Buffalo,
Wisconsin,
NY 1 4 2 6 0
Milwaukee,
WI 5 3 2 2 6
1984
Summary: The ability of hemin to stimulate estrogen synthetase (aromatase) in cultured human trophoblast cells and in cellular homogenates was investigated and compared with aromatase stimulation by dibutyryl cAMP [ ( B u ) 2 c A M P ] . Cells grown with hemin for 24 h, or homogenates incubated for 45 min with hemin, showed maximal aromatase stimulation ( 1 5 0 t o 200% o f a c t i v i t i e s in the absence of hemin) at 25 ~M a n d 0 . 1 ~M, r e s p e c t i v e l y . Aromatase stimulation in culture by 25 ~M h e m l n w a s o b s e r v e d within 4 h after hemin addition, while (Bu)2cAMP required more than 6 h. Intracellular heme and porphyrin levels were higher (160 to 185%) in 96 h (Bu)2oAMP-grown cells than control cells. ® 1985 Acad~ic Press, Inc.
Regulation estrogen
as
the
synthetase
physiological and
of
is
of
processes, clinical
P-450
of
the
enzyme
mechanism In
one
(Bu)2cAMP
plays as
in
pharmacological
its
plus
(3,4),
system,
protein
(5).
component
several
androgens,
important
role
in
and
fetal
pathological There
are
inhibition
(1,2)
relatively
few
many
development,
processes,
many
basic
of
this
studies
choriocaroinoma
h by
aromatase
Abbreviations used: (Bu)2cAMP iB~u)~ p-~uus T mM t h e o p h y l l i n e .
0006-291X/85 $1.50 Copyright © 1985 ~ A c a ~ m ~ Press, ~c. AH righ~ ~ repro~c~n m aM ~ r m rese~eK
aromatase
a process
Cytochrome of
from
reproduction
stimulate
96
an
estrogens
and
such clinical
cytochrome of
the
in
culture,
regulation.
human
theophylline over
synthesis
but
physiological
model
makes
carcinomas.
eight-to-ten-fold
regulatory
such
interest
system
of
which
(aromatase),
estrogen-dependent
studies
enzyme
P-450 in
these
dibutyryl
232
cells activity
requiring
RNA a n d
appears
to
cells
(5,6).
cAMP,
dbT
be
-
the We r e p o r t
I
mM
VOI. 127, No. 1, 1985
here
the
aromatase of
hemin
cells
in
ability in to
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of
these
to
cells.
stimulate
culture
hemin
as
a well
rapidly This
and
is
the
steroidogenic as
in
first cytochrome
cellular
KATERIALS
significantly report P-450
stimulate of
the enzyme
ability in
homogenates.
AND METHODS
~ ! $ ! ~ : The arom~tase subst~]te, [l~-3H, 4-14C] androstenedione (64.4 Ci -H, 1.7 Ci --C per mol), was prepared using the drastic alkali treatment (7) on a m~ture of [I,2"~H] androstenedione (41 Ci/mmol f r o m NEN) a n d [ 4 ~ ' C ] androstenedione (57.4 Ci/mol from NEN). The distribution of H is 77~ in the i~ position (by nmr from NEN). Hemin (Type I, bovine), protoporphyrin IX, oxalic acid, non-radioactive steroids, cytochrome c a n d NADPH w e r e p u r c h a s e d from Sigma Chemical Co. Liquiscint scintillation cocktail came from National Diagnostics. GIBCO s u p p l i e d the cell culture media and serum. Methods: Cell Culture: Human choriocarcinoma cells (JAr line) were maintained in continuous culture with growth medium changed daily, and grown in the absence or presence of dbT as previously described (8). The appropriate amount of hemin (dissolved under reduced light conditions i n 0 . 1 M NaOH, b r o u g h t to approximate pH 8 . 5 w i t h 0.1 N HC1 a n d f u r t h e r diluted with culture medium to the proper concentration) was maintained in the cell culture medium in the dark for the time periods indicated. At termination, the attached cells were washed twice using serum-free culture medium containing the appropriate amount of hemin, removed from the flask and collected by centrifugation as described (9). Enzyme and Protein Ass~x~: Frozen cell pellets were thawed and homogenized i n GP b u f f e r (5). NADPH-cytochrome c reductase activity (5) and protein content (10) were measured as described. The aromatase assay,~performed in duplicate o~ t r i p l i c a t e , was based on the transfer of "H from the substrate t o ~H_O 5 ~oncomitant with estrogen production (4), using subs~rate ( 1 0 dpm "H, 200 ng androstenedione), 0 . 5 mg NADPH a n d h o m o g e n i z e d cells (0.8 t o 2 mg p r o t e i n ) in a total volume of 1 ml. Control experiments verified substrate3saturation and linearity of the assay. The assay was conducted and H20 quantitated as described (9). We v e r i f i e d in selected samples that the apparent activation of aromatase by hemin using the -H20 assay is correlated with an increase in radiolabeled estrogen production (procedure as in (11)). ~ ~ h X ~ ! ~ _ ~ ! ! X : Cellular heme and porphyrin content were measured by the method of Granick et a!.(12) and Sassa et !~. (13), modified as described here, after washing the cells twice in 0.15 M NaC1. The cell pellet was homogenized in 0.067 M phosphate buffer, pH 7 . 4 , and aliquots mixed with 1 ml o x a l i c acid (saturated solution at 21°C) for heme measurement or extracted with 1 ml o f PM [ m i x t u r e of 1 N perchloric acid-methanol (1:1, v/v)] for porphyrin measurement. For the heme assay, samples were heated at 100°C for 30 min, 0 . 5 ml o f PM a d d e d after cooling to room temperature, and the 1,000 xg-20 min supernatant assayed. Fluorescence was measured in an Aminco-Bowman spectrofluorometer, with a red-insensitive photomultiplier tube (1P21, $4 s p e c t r a l response, 0 . 2 mm s l i t ) , a t 4 0 6 nm excitation (slit-22nm) and emissions (slit-11 nm) o f 5 7 7 - 5 7 8 nm (minimum) a n d 5 9 6 nm ( m a x i m u m ) . Control experiments showed that the 233
Vol. 127, No. 1, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
fluorescence was linearly related to the amount of protein in the assay over the range utilized. Statistical Analxsis: All experiments described here were performed at least twice. Except where otherwise noted, all assays were in duplicate or triplicate and the results reported as mean±S.D. Statistical comparisons were made using Student's t-test.
RESULTS AND D I S C U S S I O N Various control
concentrations
and
dbT-grown
activities
of
determined
(Fig.
specific to
and
about failed
10
aromatase 1).
in
the
~M f o r
to
and The
were cells
hemin
stimulation
absence
incubated in
NADPH-cytochrome
optimal
dbT-srown
affect
hemin
choriocarcinoma
significant
aromatase
of
of cells.
was
This
25
optimal
secretion
of
human
glycoprotein
hormone
u-subunit
or
progesterone
cells
unpublished
virus-transformed concentrations
18ot-
erythroleukemia above
IA
work).
.
.
!
100
.
~M w a s
.
As
.
in to
for
relative cells
concentration
control
mouse
culture
these
0.00S
gonadotropin, from
reported
the
control
hemtn
chorionic
cells toxic
(p(
specific
were for
~M f o r
h with
and
reductase
aromatase
the
(R.H.,
culture
concentration
of
hemin)
c
24
for
(14),
cells.
Friend hemin These
data
B
/
"o ~ 140i ._~ o 100~-
-
,5 10
50 100
,500 1000 5 10 Hernin Concentrotion (p.M)
-
50
100
Fig. I. Aromatase stimulation by h c m i n i n c u l t u r e d cells. (A) Choriocarcinoma c e l l s w e r e g r o w n w i t h medium c o n t a i n i n g hemin as indicated. (B) Same a s (A) e x c e p t dbT was i n c l u d e d in the medium. The I 0 0 ~ ( h e m i n - f r e e control) v a l u e s f o r a r o m a t a s e (X; p m o l / m l n / m g protein), NADPH-cytoehrome c reductasc (• ; n m o l / m l n / m g protein), and protein (e;mg/flask) w e r e f o r (A) 0 . 2 3 , 2 . 5 8 a n d 5 . 5 , r e s p e c t i v e l y , and f o r (B) 0 . 9 5 , 2 . 5 5 a n d 5 . 5 , r e s p e c t i v e l y . These data are a c c u m u l a t e d f r o m and r e p r e s e n t a t i v e of five separate determination for control cells and two f o r d b T - g r o w n c e l l s . 234
and
Vol. 127, No. 1, 1985
show
for
hemin
the
of
first
mechanism of
following
the
1 do
treatment P-450
performed
in
mechanism
of
insertion
into
mechanism
of
trations that
action.
with
agents
(15)
or
that
in
cells
extent
of
aromatase
cultured
cells
occurs
at
0.1
together
suggest
values
in
aromatase
the
absence
cytochrome
by
a
Both
hemin)
P-450
in
results
these
that
sets
of
aromatase
by
activation
suggests
that
cells
is
to
hemin
as
about in
the
(16),
fractions,
We t e s t e d
activation
of
of
regarded
The
tissues
tissues
various
X ~ -
activates
of
hepatic
formation
(17,18).
the
stimulation
subcellular
incubating
extent
the
in
generally
~M h e m i n .
the
of
the
and/or
~n
similar
Furthermore,
of
of
non-hepatic
P-450
a
mechanism.
studies
promote
is
by
hemin
and
identification
untreated
activation
activation
that
stimulation
cells
hydzoxylation,
homogenates
homogenized
knowledge,
cultured
previous
apocytochzome
hemin
our
permit
drug
with
hemin
the
in
not
Since
tissue
of
enzyme.
P-450-dependent
apocytochrome were
in
Fig.
best
enzyme
P-450
heme
cytochzome
P-450
the
cytochrome
results of
to
time,
a cytochrome
steroidogenic The
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in seen data
an
be
X~!
this concenFig.
2
with taken
insertion
(150
to
200%
of
30
to
50% o f
apocytochzome
form.
o
200
§ "5 150
g ~
100,
~
50
o
o o
,
I
200
,
I
400
,
I
600
,
I
800
,
I
1000
[Heroin] (nM) Fig. 2. Aromatase stimulation by hemin in cellular homogenates. Control choriocarcinoma cells were homogenized and incubated with hemin as indicated a n d 1 . 5 mM EDTA f o r 45 m i n a t 3 7 ° C i n f l a s k s protected from light. The aromatase essay was initiated by adding substrate a n d NADPH f o l l o w e d by incubation a t 3 7 ° C f o r 32 m i n . The control (100%) aromatase activity was 0.055 pmol/min/mg protein.
235
show
the
Vol. 127, No. 1, 1 9 8 5
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
350
/
= 300 ~6 250 200 ~150 o o 100 '< 5o
O0
I
I
2
i
I
4
,
I
/
J
6 Time (hr)
"
2
~4
Fig. 3. Time c o u r s e of h e m i n - and d b T - s t i m u l a t i o n of aromatase in culture. Cells were grown in medium alone (control), medium with 25 ~M h e m i n ( . ) , o r m e d i u m w i t h dbT ( [] ) . Aromatase specific activities i n h e m i n - a n d dbT - c o n t a i n i n g medium were divided by the specific activity of control cells at each time point. The a v e r a g e NADPH-cytochrome c reductase activity or protein content throughout t h e 24 h w e r e n o t s i g n i f i c a n t l y different in control or treated cells. The c o n t r o l aromatase activities (pmol/min/mg protein) at zero time averaged 0.25-+ 0.012 (n=10), increasing to 0.46 + 0.006 (n=4) at 6 h before declining to 0.3+0.005 ( n = 4 ) a t 24 h .
To
determine
stimulation
of
aromatase (Fig. but
3). not
48
the
aromatase
At in
h.
4
and
control
By 2 4 in
h
cells
grown
dbT.
The when
higher relative
in
aromatase
levels to
of control
25
FM h e m i n
aromatase 24
sought
and
to
activity and
dbT
activity
in
significantly
in
cells
hemin-grown than
cells, the
value
cells
from
24
exceeded
cells. if
by
intracellular
of
time
without
that
only
comparing
stimulated observed
236
of
cultured
dbT-growu
dbT
cells.
in
course
a maximum
to
porphyrin
time
higher
reaching
determine
suggest is
well-documented the
pM h e m i n - g r o w n
periods I
the
by
activity
responsive
Table
in
compared
(p<0.00$),
and
heme
we
was
various
data
dbT,
cells,
are
role
by
aromatase
we
for
a
6 h,
the
control
levels
plays
cells
Furthermore, porphyrin
by
dbT-grown
in
value
dbT,
heme
stimulation
activity to
if
by
heme
these
levels
(control)
after 8-to in
96
h
or
in
with
growth
10-fold
dbT-grown
and
(5), cells
with were
Vol. 1 2 7 , No. 1 , 1 9 8 5
Table
1.
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
Porphyrin
a n d heme
content
of
choriocarcinoma
Porphyrin time
control
cells
Heme
dbT-grown
control
dbT-grown
pmol/mg
pmol/mg prot.
prot.
8.15 ± 2.28
13.45+_ 1 . 2 3 *
Experiment
I
96 h
0.51
+_0.15
0.94 ± 0.42
Experiment
II
48
h
1.55
+_0.31
1 . 2 5 -+ 0 . 0 9 8
11.6
+_ 0 . 7 1
13.3_+ 0 . 4 7 *
72 h
0.92
±0.07
1.2
11.3
+_ 1 . 3 5
11.9+_ 0 . 9 6
96
0.72
+_0.1
1.34 + 0.11"
h
± 0.063*
8 . 4 3 _+ 0 . 5 4
1 3 . 3 _+ 0 . 7 7 *
C e l l s w e r e g r o w n i n m e d i u m a l o n e o r w i t h dbT f o r t h e i n d i c a t e d time, end then washed and collected as d e s c r i b e d . Aliquots of homogenized cells were a s s a y e d f o r heme a n d p o r p h y r i n content end protein concentration. Porphyrin was undetectable in both growth medium and undiluted serum. Eeme l e v e l s w a s 1 . 5 7 pM i n 10% s e r u m . The t o t a l heme c o n t e n t of the growth medium per flask divided by the total cellular protein attached to the flask i s a b o u t 2 . 4 nmol heme/mg p r o t e i n . • v (
0.005
relative
Provided 10
to
20%
of
consistent involve the to
i~
that the
heme
intracellular
~K~
the
regulation
of
the that
apocytochrome significant culture
enzyme.
of
is
aromatase and
i~
heme
in
vitro.
Since
1
these
this is
data
dbT
less
than
are by
heme
dbT
supply
apocytochrome
unique
limiting
by
are
stimulation
aromatase
assumptions
stimulation
levels
intracellular
into
--
I
aromatase
heme
present
time.
P-450
the
Implicit
intracellular
P-450
same
levels that
insertion
at
eytochrome
hypothesis
assumptions
in
aromatase
cells
the
activate
hemin
control
with
subsequent
and
to
that
supported
optlmal
are
the
aromatase by
the
concentrations
stimulates
and P-450
hypothesis and
may
aromatase
rapid of to
a
Direct estimates of the total cytochrome P-450 in homogenized, subcellular-fraetionated JAr cells w e r e 3 a n d 13 p m o l / m g protein in the low-speed pellet from control and dbT-grown cells, respectively, with undetectable levels ((2 pmol/mg protein) in the eel1 homogenate and microsomal fractions (8). Aromatase cytochrome P-450 constitutes only a fraction of the total cytochrome P-450. By extrapolating cytochrome P-450 levels from human placental microsomes to JAr cells based on the relative aromatase specific activity, we estimate aromatase cytochrome P-450 levels in JAr cells at about 0.1 and 1 pmol/m 8 protein in control and dbT-grown cells, respectively. 237
Vol. 127, No. 1, 1985
much g r e a t e r in
the
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
extent
mechanism
of
than
hemin,
additional
factors
must
be
involved
dbT a c t i o n .
Acknowledgments: We t h a n k Mr. 7 . K u r t z , Ms. A. R u c k e r t , M r s . M. R i n k e a n d Ms. M.McAsey f o r t e c h n i c a l assistance and Mrs. U.Brunn a n d Mr. 7 . S t a m o s f o r a s s i s t a n c e with the manuscript. Supported by U.S.P.H.S.Grant HD 1 6 5 9 3 .
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7r.,
and Gunsalus,
I.C.