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5′-Deoxypyridoxal inhibition of glucocorticoid receptor binding in HeLa S3 cells and rat thymocytes
Vol.
92, No.
Jonwary
BIOCHEMICAL
1, 1980
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
15, 1980
155-162
5'-DEOXYPYRIDOXALINHIBITION OF GLUCOCORTICOID RECEPTOR BINDING IN HeLa S3 CELLS AND RAT THYMOCYTES Jane Margaret
John W. Thanassi
O'Brien,
and John A.Cidlowski
Department of Biochemistry University of Vermont Burlington, Vermont 05405
Received
November
19,1979 Summary
Recent evidence suggests a possible role for pyridoxal 5'-phosphate, the active physiological form of vitamin 66, in steroid hormone action (l-3). We now report that 5'-deoxypyridoxal, a synthetic vitamin B causes a rapid and complete loss of dexamethasone receu i or antagonist, binding in cytosol preparations in whole HeLa S3 cells or in rat thymocytes. This effect is concentration and time dependent, and is specific for 5'-deoxypyridoxal. In whole cell incubations of either HeLa S3 cells or rat thymocytes at 37'C, a 50% reduction in [3H]dexamethasone binding was observed in the presence of 0.25 mM 5'-deoxypyridoxal. Cytotoxicity was not evident in rat thymocytes or HeLa S cells incubated with 3.0mM or l.OmM 5'-deoxypyridoxal respective ?y. Pyridoxal 5'-phosphate, 5'-deoxypyridoxine and 5'-deoxypyridoxamine were ineffective in causing a loss of steroid receptor binding. These studies suggest that 5'-deoxypyridoxal may be an effective non-steroidal compound which can effect the binding of glucocorticoids to specific receptor proteins in whole cells.
Introduction Steroid
antagonists
provide
mechanism
of hormone-receptor
potential
chemotherapeutic
dependent
cancers.
steroidal between procedures
antagonists large
little
have not
particularly
(6-9). been fully
probing they
in the
useful
antagonists
hormonal
activity,
doses
for
In addition,
are of particular
Non-steroidal
and mineralocorticoids antagonists
agents,
pharmacological (5).
tools
binding.
Clinically
at low concentrations,
invaluable
must
exhibit
of hormones
and major
Analogous
of interest
known
non-steroidal
for
as
of hormonehigh
and low toxicity providing
are
are
treatment
interest,
antagonists
the basic
potency (4).
Non-
an alternative ablative estrogens,
surgical androgens
glucocorticoid
developed. 0006-291X/30/010155-08$01.00/0
155
Copyright 0 1980 by Academic Press, Inc. A II rights oJ reproduction in any form reserved.
Vol.
92, No.
Evidence action
suggests
of hormone
5'-deoxypyridoxal from specific were
BIOCHEMICAL
1, 1980
that
chosen,
receptor
causes
a rapid
as they
coid
(12,73).
able
as a mammalian receptors
5'-deoxypyridoxal at Z"C, A possible
and for role
which
line,
nuclear
with
for for
receptor
of 5'-deoxypyridoxal
by blocking
cell
cortexolone,
types.
from a cervical
contain
high
affinity (14).
receptor
in whole
Rat thymocytes
receptors
derived
cytoplasmic
that
a steroidal
glucocorticoids
binding
the
of [3H]dexamethasone
glucocorticoid
which
specific
was observed
loss
from two different
HeLa S3 cells
are
act
COMMUNICATIONS
We have demonstrated
and complete
in studies
cell
RESEARCH
antagonists
have well-characterized
employed
chosen
BIOPHYSICAL
(7,10,11).
proteins
been previously antagonist
some hormone
at its
receptor
AND
cell
as a glucocorticoid
and have
glucocorticarcinoma
were
saturThe effect
in cytosol preoarations antagonist
of
preparations at 37°C. is
suggested.
Materials
and Methods
Thymus tissue was obtained from male Sprague-Dawley rats which were adrenalectomized 4-6 days prior to sacrifice. Thymus cell suspensions were prepared in Krebs-Ringer bicarbonate buffer with 10 mM glucose, pH 7.4 (KRBG), equilibrated with 95% 02/5% CO2 (15). Cells were used at a cytocrit of 0.1-0.2 ml of packed cells per ml of cell suspension. The cytocrit was determined by the microhematocrit method. HeLa S3 cells were grown to confluency as monolayer cultures in Joklik's Minimum Essential Medium (Grand Island Biological Company) suoplemented with 7 l/2% fetal calf serum and 2 mM glutamine in a humidified 5% oxygen/95% air atmosphere at 37°C. Cells were harvested by treatment with versene, washed with 2-3 volumes of unsup lemented Joklik's medium and resuspended to a final . concentration of l-3 x 10 P viable cells/ml. Cell viability was determined in a hemacytometer using exclusion of Trypan Blue as a criterion. [6,7-3HlDexamethasone (36.0 Ci/mmol) was purchased from New England Nuclear and stored at 4'C in benzene:ethanol (9:1, v:v). Solvent was evaporated prior to the addition of cell suspensions. Stock solution of unlabeled dexamethasone (Steraloids) was orepared at a concentration of 1 x 10-4 M in KRBG buffer. 5'-Deoxypyridoxal was prepared by the method of Muhlradt and Snell (16). 5'-Deoxypyridoxine and 5'-deoxypyridoxamine were prepared by catalytic hydrogenolysis of phosphorylated precursors (17). Stock solutions (50 mM)wereprepared in 1.5 mM MgC12, adjusted to pH 7.0 with KOH, and frozen until use. Oextran-coated charcoal (1.0% Norit, 0.1% dextran) was prepared in 1.5 mM Other chemicals were reagent grade and were MgC12 and stored at 4°C. obtained from Sigma or Fisher. Aliquots of thymus cell 2 x 10-B M [3H]dexamethasone
or HeLa S3 cell suspension alone or in the presence
156
were incubated with of a lOO-fold molar
Vol.
92,
No.
BIOCHEMICAL
1, 1980
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
excess of unlabeled dexamethasone for 1 l/2-2 h at 2°C. The difference in binding values obtained for cells incubated in the presence and absence of unlabeled dexamethasone represented the saturable receptor binding fraction. Thymocyte nuclei were prepared and nuclear receptor binding was measured using the hypotonic lysis technique of Munck and Wira (18). HeLa S3 cell nuclear receptor binding was measured by the freeze-thaw hypotonic lysis procedure Thymocyte cytoplasmic fractions were described by Sibley and Tomkins (19). prepared by hypotonic lysis in 1.5 mM MgC12 (18). HeLa S3 cytoplasmic fractions were prepared by suspending the cells in l-3 ml of 1.5 mM MgC12, and homogenizing with three-10 set bursts of a chilled Tekmar Ultra-Turrax homogenizer apparatus. Cellular debris was removed by a 1 min centrifugation at 10,000 x g. The supernatant represented the cytoplasmic receptor fraction or "cytosol". Results Fig. to
1 shows the
nuclear
Incubation
receptor
in
of cells
in the
deoxypyridoxal methasone
binding
5'-Deoxypyridoxal dexamethasone
effect
for
of 5'-deoxypyridoxal
whole
HeLa
S3 cells
presence
(A)
nuclear
receptor
concentrations receptor
binding
and
of millimolar
30 min at 37°C caused to
on [3H]dexamethasone
both
rat
thymocytes
concentrations
a decrease
in
S3 cells
and
[3H]dexathymus
a 50% reduction
HeLa S3 cells
(8).
of 5'-
of saturable
HeLa
of 0.2 mM caused in both
whole
binding
cells.
in nuclear
and thymocytes.
At 5'-
f Lx u w
B THYWS -----
0.5 - IO
3.0
5.0
S’-DEOXYPYRIDOXAL S’-OEOXYPYRIDOXAL
_----
---
0.5 o.s-
-_ - ---_
1.0
2.0
- *
.I003
3.0
(mM)
Figure #l. The influence of 5'-deoxypyridoxal treatment of whole cells on nuclear dexamethasone receptor Binding. HeLa S3 cells or rat thymocytes were incubated with ei her 2 x lo- M [ Hldexamethasone alone or [3H]dexamethasone ~1~s 2 x lo- h M unlabeled dexamethasone for 30 min at 370C. The cells were then treated with the concentrations of 5'-deoxyDyridoxa1 indicated for an additional 30 min at 37'C. Nuclear binding was measured using a hypotonic lysis assay (see Methods). The data shown represent the saturable binding fractions (mean + S.E.; 2 determinations) calculated by subtracting binding values obtained in the presence of 2 x 10s6 M unlabeled dexamethasone. S'-DeoxyDyridoxal did not alter the level of nonsaturable binding. Cells were cooled to Z°C immediately after incubation and viability determined one-half hour after incubation was CornDieted.
,157
Vol.
92, No.
1, 1980
deoxypyridoxal
similar
to less controls.
binding cell
death,
were
not affected
compound thymus
cell
which cell
similar
(data
whether
5'-deoxypyridoxal
viability
was determined.
incubation
by
reduced
with
5'-deoxypyridoxal
1B).
(4.5
HeLa S3 cell
mM) decreased
reduction
in nuclear
incubated
with
prior
to equilibration
HeLa S3 cell
of time
of [3H]dexamethasone and thymus
cytosol
preparations
were
deoxypyridoxal.
with c3H]-
receptor
was a consequence cell
of
viabilities
at concentrations
binding.
No decrease
of the in
concentrations
was similarly
as
unaffected
at
High
concentrations
of 5'-deoxy-
viability
by 70%.
Thus a = 98%
receptor
binding
occurs
a concentration
of incubation
in whole
which
cells
does not affect
binding
on the 5'-deoxypyridoxal-induced
to isolated
preparations incubated
is shown for
cytoplasmic
receptor
in Fig 2.
Cytoplasmic
60 min at 2°C in the presence
A first-order
exponential
decrease
sone was observed,
with
k-l=
2.24
x 10-2
methasone
complexes
(Fig
2A) and k-,=2.31
receptor binding
receptor complexes in the
(Fig
presence
Heta S3 and thymus
corrected
for
ing which
occurs
loss
and
viability. The effect
for
binding
with
HeLa S3 and thymus
viability
1 mM 5'-deoxypyridoxal,
thymocytes
glucocorticoid
treatment
of ~1 mM.
glucocorticoid
recentor
shown).
at 5'-deoxypyridoxal
concentrations
pyridoxal
or rat
not
COMMUNICATIONS
HeLa S3 cells
cells
receptor
was observed
both
5'-deoxyoyridoxal
glucocorticoid
viability
for
the decreased
following
as 3.2 mM (Fig
cell
results
RESEARCH
dexamethasone
found
of HeLa S3
to determine
observed
BIOPHYSICAL
of 5'-deoxypyridoxal
gave
In an effort
2% of the binding
Incubation
concentrations
dexamethasone
high
than
AND
of ~1 mM, saturable
concentrations
was reduced thymocyte
BIOCHEMICAL
non-saturable
The half-life
28).
cytoplasmic
binding
and for
x 10
of added
158
receptor of 5 mM 5'[3H]dexamethadexathymocyte
of ["Hldexamethasone 31 min and t1,2=30 Values
of added
After
min
have been
of [3H]dexamethasone
5'-deoxypyridoxal.
in the absence
HeLa S3
-2 min -1 for
respectively. loss
from
HeLa S3 cytoplasmic
was tl,2=
receptors,
binding
for
of loss
of 5'-deoxypyridoxal
in the absence
of [3H]dexamethasone
min-'
in bound
loss
bind60 min,
5'-deoxypyridoxal
Vol.
92,
No.
BIOCHEMICAL
1, 1980
f
AND
A 20
BIOPHYSICAL
COMMUNICATIONS
B THYWS
HsLo~ I 60
40
RESEARCH
t 20
40
60
MIWTES
Figure ii2. 1-he influence of 5'-deoxyoyridoxal on dexamethasone recentor binding in isol ated cytosols. Isolated HeLa S3 cell; or thymocytes were incubated with loM [SHldexamethasone for 2 h at 7 C. Aliquots (0.9 ml) of cytosol were brought to 5 NM 5'-deoxynyridoxal with the addition of 0.1 ml of 50 ml4 stock solution (PH 7.6). Controls received an equivalent amount of 1.5 mM MgC12., At intervals thereafter 100 ~1 aliquots of cytosol were removed, treated with 100 ~1 of dextran-coated charcoal for 5 min and centrifuged at 10,000 x g for 1 min. Samples of the supernates were placed into scintillation vials for the determination of radioactivity. The data was nlotted as the % of the control [SH]dexamethasone binding observed in 5'deoxyoyridoxal-treated cytosol. [3H]Dexamethasone binding in control cytosols is indicated by open circles. Data from three separate exneriments are plotted
only
represented
thymus
5-10%
cytosols.
show
that
whole
These
at at
Several
at 2°C. little
decrease
similar
loss
to
control
those
(37°C)
and
next
examined
HeLa
S3 and
represented
glucocorticoid
the of
were
thymocyte
rat
concentration
in
importance by
decrease
analogs
in
in
Fig
receptor
binding
isolated
cytoplasmic
of
on
the
In
cytosol
the
5 mM were
of failure steroid-receptor
the of
of incubated
of
added
1, in
effect
receptors
to
thymus
bound
with
alcohol In
159
cytosol
for
5'-deoxypyridoxain
5 mM 5'-deoxypyridoxal,
group
addition,
was of
a
observed
after
5'-deoxypyridoxal or
amine the
3).
5'-deoxy-
[3tt]dexamethasone
[3H]dexamethasone
corresponding binding.
bound
on (Fig
or
and
saturable
C4'-carboxaldehyde the
their
cytoplasmic
5'-deoxypyridoxine,
amount
amount
for
5'-deoxypyridoxamine
5'-phosphate,
effect
six-fold
indicated
in
with
temperatures
Pyridoxal
preparations.
The
binding
conjunction
5'-deoxypyridoxine,
a final
cytosol
2 h.
can
binding
pyridoxal
had
in
5'-deoxypyridoxal
5'-phosphate,
mine
data,
initial
2°C.
Pyridoxal
at
total
physiological
[3H]dexamethasone
2 hr
the
5'-deoxypyridoxal
cells
fractions
of
to
presence
is effect
a of
the
Vol. 92, No. 1, 1980
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
too I-
Figure if3. The specificity of 5'-deoxypyridoxal action on cytoplasmic dexamethason receptor. Thymocytes were incubated at 2OC for 2 h with either 2 x 10m8 M [ 5 Hldexanethasone alone or [3Hldexamethasone plus 2 x 10e6 M unlabeled dexamethasone. Cytosols were prepared and incubated with 5 mM of the indicated compounds at 2OC for an additional 2 h. Receptor binding was measured with the dextran-coated charcoal assay and saturable binding values (mean i S.E., 2 determinations) are plotted.
apolar
methyl
substituent
glucocorticoid charged
at
receptor C5'
phosphate
receptor
binding.
specific
manner
appears
These
to
is
essential
for
5'-phosphate
ineffective
data.indicate
reduce
be
Pyridoxal
binding.
group,
to
C5'
that
glucocorticoid
with
in causing
a loss
5'-deoxypyridoxal receptor
the
reduction
a highly
of steroid acts
binding
in
in
in
these
a highly
prepara-
tions.
Discussion We have Bg analog, receptor
demonstrated decreases
complexes
in
isolated
cytoplasmic
of
[3H]dexamethasone
that the
in
5'-deoxypyridoxal,
binding
of
S3 cells
HeLa
fractions binding
dexamethasone and at
was
a potent
found
rat 2°C. for
160
to
synthetic
nuclear
thymocytes
--in
glucocorticoid vitro
A rapid
and
nuclear
receptor
vitamin
nearly
at
37°C
and
complete complexes
loss in
whole
Vol.
92. No.
HeLa
BIOCHEMICAL
1. 1980
S3 cells
and
rat
5'-deoxypyridoxal, mocyte
with
cytosol
be quite as
from than
at
specific
for
in
2"C,
5'-phosphate,
RESEARCH
37°C
cell
in
the
viability.
similar
dissociation
were
control
producing
at in
complexes
found
BIOPHYSICAL
incubated
alteration
receptor
those
pyridoxal
no
preparations
dexamethasone greater
thymocytes
AND
presence
of
1 mM
In
S,
and
HeLa
rates
calculated,
cytosols.
COMMUNICATIONS
of
approximately
in
5'-deoxypyridoxal
appears
glucocorticoid and
[3H]-
20-fold
5'-Deoxypyridoxal
reductions
bound
thy-
receptor
to binding,
5'-deoxypyridoxamine
were
ineffective. 5'-Deoxypyridoxal decrease
the
in
cells.
whole
sult
of
its
binding
of
with its
the
be
a non-steroidal
steroid
B6 receptor
as
to
effects
vitamin
potential
to
glucocorticoids
Whether inherent
interaction theless,
appears
of
antagonist remains
a non-steroidal
compound
specific
receptor
5'-deoxyoyridoxal action to
be
due
established.
glucocorticoid
can
proteins are
or
which
to
the
re-
a direct Never-
antagonist
is
suggested.
Acknowledgements -_-Supported
in
part
by
NIH
grants
AM 20892
and
AM 20762.
References ___1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13.
Cidlowski, J.A. and Thanassi, J.W. (1978) Biochem. Biophys. Res. Commun. 82, 1140-1146. Cidlowski, J.A. and Thanassi, J.W. (1979) Biochemistry 18, 2378-2384. Cake, M.H., DiSorbo, D.M. and Litwack, G. (1978) J. Biol. Chem. 253, 4886-4891. Mainwaring, W.I.P. (1975) in Vitamins and Hormones (Munson, P.L., Glover, J Diczfalusy, E. and Olsen, R.E., eds) Vo1.33, PP. 223-245, Academic Pi&s, New York. Carbone, P.P. (1975) Ann. Intern. Med. 83, 730-731. Neri, R. and Monahan, M. (1972) Invest. Urol. 10, 123-130. Marver, D., Stewart, J., Funder, J., Feldman, D., and Edelman, I. S. (1974) Proc. Natl. Acad. Sci. 71, 1431-1435. Terenius, L. (1971) Eur. J. Cancer 7, 57-64. Horwitz, K.B. and McGuire, W.L. (1978) J. Biol. Chem. 253, 8185-8191. Korennan, S.G. (1968) Endocrinology 87, 1119-1123. Munck, A. and Wira, C. (1971) in Advances in the Biosciences (Raspe, G. ed.) Vol 7, Perganon Press, Vieurg pp. 301-330. Kaiser, N., Milholland, R., Turnell, R.W. and Rosen, F. (1972) Biochem. Biophys. Res. Commun. 49, 516-521. Turnell, R.W., Kaiser, N., Milholland, R.J. and Rosen F. (1974) 249, 1133-1138.
161
Vol.
92, No.
14.
;65: K:
19.
1, 1980
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Melnykovych, G. and Bishop, C.F. (1969) Biochem. Biouhys. Acta. 177, 579-585. Munck, A. (1968) J. Biol. Chem. 243, 1039-1042. Mijhlradt, D.F. and Snell, E.E. (1967) J. Med. Chem. 10, 129-130. Cunningham, W.C., and Thanassi, J.W. (1979) Experientia 35, 451-452. Munck, A. and Wira, C. (1975) in Methods in Enzymology (O'Malley, B.W. and Hardman, J., eds.) Vol. 36, Academic Press, New York, pp. 255-266. Sibley, C.H. and Tomkins, G.M. (1974) Cell 2, 221-227.
162
92, No.
Jonwary
BIOCHEMICAL
1, 1980
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
15, 1980
155-162
5'-DEOXYPYRIDOXALINHIBITION OF GLUCOCORTICOID RECEPTOR BINDING IN HeLa S3 CELLS AND RAT THYMOCYTES Jane Margaret
John W. Thanassi
O'Brien,
and John A.Cidlowski
Department of Biochemistry University of Vermont Burlington, Vermont 05405
Received
November
19,1979 Summary
Recent evidence suggests a possible role for pyridoxal 5'-phosphate, the active physiological form of vitamin 66, in steroid hormone action (l-3). We now report that 5'-deoxypyridoxal, a synthetic vitamin B causes a rapid and complete loss of dexamethasone receu i or antagonist, binding in cytosol preparations in whole HeLa S3 cells or in rat thymocytes. This effect is concentration and time dependent, and is specific for 5'-deoxypyridoxal. In whole cell incubations of either HeLa S3 cells or rat thymocytes at 37'C, a 50% reduction in [3H]dexamethasone binding was observed in the presence of 0.25 mM 5'-deoxypyridoxal. Cytotoxicity was not evident in rat thymocytes or HeLa S cells incubated with 3.0mM or l.OmM 5'-deoxypyridoxal respective ?y. Pyridoxal 5'-phosphate, 5'-deoxypyridoxine and 5'-deoxypyridoxamine were ineffective in causing a loss of steroid receptor binding. These studies suggest that 5'-deoxypyridoxal may be an effective non-steroidal compound which can effect the binding of glucocorticoids to specific receptor proteins in whole cells.
Introduction Steroid
antagonists
provide
mechanism
of hormone-receptor
potential
chemotherapeutic
dependent
cancers.
steroidal between procedures
antagonists large
little
have not
particularly
(6-9). been fully
probing they
in the
useful
antagonists
hormonal
activity,
doses
for
In addition,
are of particular
Non-steroidal
and mineralocorticoids antagonists
agents,
pharmacological (5).
tools
binding.
Clinically
at low concentrations,
invaluable
must
exhibit
of hormones
and major
Analogous
of interest
known
non-steroidal
for
as
of hormonehigh
and low toxicity providing
are
are
treatment
interest,
antagonists
the basic
potency (4).
Non-
an alternative ablative estrogens,
surgical androgens
glucocorticoid
developed. 0006-291X/30/010155-08$01.00/0
155
Copyright 0 1980 by Academic Press, Inc. A II rights oJ reproduction in any form reserved.
Vol.
92, No.
Evidence action
suggests
of hormone
5'-deoxypyridoxal from specific were
BIOCHEMICAL
1, 1980
that
chosen,
receptor
causes
a rapid
as they
coid
(12,73).
able
as a mammalian receptors
5'-deoxypyridoxal at Z"C, A possible
and for role
which
line,
nuclear
with
for for
receptor
of 5'-deoxypyridoxal
by blocking
cell
cortexolone,
types.
from a cervical
contain
high
affinity (14).
receptor
in whole
Rat thymocytes
receptors
derived
cytoplasmic
that
a steroidal
glucocorticoids
binding
the
of [3H]dexamethasone
glucocorticoid
which
specific
was observed
loss
from two different
HeLa S3 cells
are
act
COMMUNICATIONS
We have demonstrated
and complete
in studies
cell
RESEARCH
antagonists
have well-characterized
employed
chosen
BIOPHYSICAL
(7,10,11).
proteins
been previously antagonist
some hormone
at its
receptor
AND
cell
as a glucocorticoid
and have
glucocorticarcinoma
were
saturThe effect
in cytosol preoarations antagonist
of
preparations at 37°C. is
suggested.
Materials
and Methods
Thymus tissue was obtained from male Sprague-Dawley rats which were adrenalectomized 4-6 days prior to sacrifice. Thymus cell suspensions were prepared in Krebs-Ringer bicarbonate buffer with 10 mM glucose, pH 7.4 (KRBG), equilibrated with 95% 02/5% CO2 (15). Cells were used at a cytocrit of 0.1-0.2 ml of packed cells per ml of cell suspension. The cytocrit was determined by the microhematocrit method. HeLa S3 cells were grown to confluency as monolayer cultures in Joklik's Minimum Essential Medium (Grand Island Biological Company) suoplemented with 7 l/2% fetal calf serum and 2 mM glutamine in a humidified 5% oxygen/95% air atmosphere at 37°C. Cells were harvested by treatment with versene, washed with 2-3 volumes of unsup lemented Joklik's medium and resuspended to a final . concentration of l-3 x 10 P viable cells/ml. Cell viability was determined in a hemacytometer using exclusion of Trypan Blue as a criterion. [6,7-3HlDexamethasone (36.0 Ci/mmol) was purchased from New England Nuclear and stored at 4'C in benzene:ethanol (9:1, v:v). Solvent was evaporated prior to the addition of cell suspensions. Stock solution of unlabeled dexamethasone (Steraloids) was orepared at a concentration of 1 x 10-4 M in KRBG buffer. 5'-Deoxypyridoxal was prepared by the method of Muhlradt and Snell (16). 5'-Deoxypyridoxine and 5'-deoxypyridoxamine were prepared by catalytic hydrogenolysis of phosphorylated precursors (17). Stock solutions (50 mM)wereprepared in 1.5 mM MgC12, adjusted to pH 7.0 with KOH, and frozen until use. Oextran-coated charcoal (1.0% Norit, 0.1% dextran) was prepared in 1.5 mM Other chemicals were reagent grade and were MgC12 and stored at 4°C. obtained from Sigma or Fisher. Aliquots of thymus cell 2 x 10-B M [3H]dexamethasone
or HeLa S3 cell suspension alone or in the presence
156
were incubated with of a lOO-fold molar
Vol.
92,
No.
BIOCHEMICAL
1, 1980
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
excess of unlabeled dexamethasone for 1 l/2-2 h at 2°C. The difference in binding values obtained for cells incubated in the presence and absence of unlabeled dexamethasone represented the saturable receptor binding fraction. Thymocyte nuclei were prepared and nuclear receptor binding was measured using the hypotonic lysis technique of Munck and Wira (18). HeLa S3 cell nuclear receptor binding was measured by the freeze-thaw hypotonic lysis procedure Thymocyte cytoplasmic fractions were described by Sibley and Tomkins (19). prepared by hypotonic lysis in 1.5 mM MgC12 (18). HeLa S3 cytoplasmic fractions were prepared by suspending the cells in l-3 ml of 1.5 mM MgC12, and homogenizing with three-10 set bursts of a chilled Tekmar Ultra-Turrax homogenizer apparatus. Cellular debris was removed by a 1 min centrifugation at 10,000 x g. The supernatant represented the cytoplasmic receptor fraction or "cytosol". Results Fig. to
1 shows the
nuclear
Incubation
receptor
in
of cells
in the
deoxypyridoxal methasone
binding
5'-Deoxypyridoxal dexamethasone
effect
for
of 5'-deoxypyridoxal
whole
HeLa
S3 cells
presence
(A)
nuclear
receptor
concentrations receptor
binding
and
of millimolar
30 min at 37°C caused to
on [3H]dexamethasone
both
rat
thymocytes
concentrations
a decrease
in
S3 cells
and
[3H]dexathymus
a 50% reduction
HeLa S3 cells
(8).
of 5'-
of saturable
HeLa
of 0.2 mM caused in both
whole
binding
cells.
in nuclear
and thymocytes.
At 5'-
f Lx u w
B THYWS -----
0.5 - IO
3.0
5.0
S’-DEOXYPYRIDOXAL S’-OEOXYPYRIDOXAL
_----
---
0.5 o.s-
-_ - ---_
1.0
2.0
- *
.I003
3.0
(mM)
Figure #l. The influence of 5'-deoxypyridoxal treatment of whole cells on nuclear dexamethasone receptor Binding. HeLa S3 cells or rat thymocytes were incubated with ei her 2 x lo- M [ Hldexamethasone alone or [3H]dexamethasone ~1~s 2 x lo- h M unlabeled dexamethasone for 30 min at 370C. The cells were then treated with the concentrations of 5'-deoxyDyridoxa1 indicated for an additional 30 min at 37'C. Nuclear binding was measured using a hypotonic lysis assay (see Methods). The data shown represent the saturable binding fractions (mean + S.E.; 2 determinations) calculated by subtracting binding values obtained in the presence of 2 x 10s6 M unlabeled dexamethasone. S'-DeoxyDyridoxal did not alter the level of nonsaturable binding. Cells were cooled to Z°C immediately after incubation and viability determined one-half hour after incubation was CornDieted.
,157
Vol.
92, No.
1, 1980
deoxypyridoxal
similar
to less controls.
binding cell
death,
were
not affected
compound thymus
cell
which cell
similar
(data
whether
5'-deoxypyridoxal
viability
was determined.
incubation
by
reduced
with
5'-deoxypyridoxal
1B).
(4.5
HeLa S3 cell
mM) decreased
reduction
in nuclear
incubated
with
prior
to equilibration
HeLa S3 cell
of time
of [3H]dexamethasone and thymus
cytosol
preparations
were
deoxypyridoxal.
with c3H]-
receptor
was a consequence cell
of
viabilities
at concentrations
binding.
No decrease
of the in
concentrations
was similarly
as
unaffected
at
High
concentrations
of 5'-deoxy-
viability
by 70%.
Thus a = 98%
receptor
binding
occurs
a concentration
of incubation
in whole
which
cells
does not affect
binding
on the 5'-deoxypyridoxal-induced
to isolated
preparations incubated
is shown for
cytoplasmic
receptor
in Fig 2.
Cytoplasmic
60 min at 2°C in the presence
A first-order
exponential
decrease
sone was observed,
with
k-l=
2.24
x 10-2
methasone
complexes
(Fig
2A) and k-,=2.31
receptor binding
receptor complexes in the
(Fig
presence
Heta S3 and thymus
corrected
for
ing which
occurs
loss
and
viability. The effect
for
binding
with
HeLa S3 and thymus
viability
1 mM 5'-deoxypyridoxal,
thymocytes
glucocorticoid
treatment
of ~1 mM.
glucocorticoid
recentor
shown).
at 5'-deoxypyridoxal
concentrations
pyridoxal
or rat
not
COMMUNICATIONS
HeLa S3 cells
cells
receptor
was observed
both
5'-deoxyoyridoxal
glucocorticoid
viability
for
the decreased
following
as 3.2 mM (Fig
cell
results
RESEARCH
dexamethasone
found
of HeLa S3
to determine
observed
BIOPHYSICAL
of 5'-deoxypyridoxal
gave
In an effort
2% of the binding
Incubation
concentrations
dexamethasone
high
than
AND
of ~1 mM, saturable
concentrations
was reduced thymocyte
BIOCHEMICAL
non-saturable
The half-life
28).
cytoplasmic
binding
and for
x 10
of added
158
receptor of 5 mM 5'[3H]dexamethadexathymocyte
of ["Hldexamethasone 31 min and t1,2=30 Values
of added
After
min
have been
of [3H]dexamethasone
5'-deoxypyridoxal.
in the absence
HeLa S3
-2 min -1 for
respectively. loss
from
HeLa S3 cytoplasmic
was tl,2=
receptors,
binding
for
of loss
of 5'-deoxypyridoxal
in the absence
of [3H]dexamethasone
min-'
in bound
loss
bind60 min,
5'-deoxypyridoxal
Vol.
92,
No.
BIOCHEMICAL
1, 1980
f
AND
A 20
BIOPHYSICAL
COMMUNICATIONS
B THYWS
HsLo~ I 60
40
RESEARCH
t 20
40
60
MIWTES
Figure ii2. 1-he influence of 5'-deoxyoyridoxal on dexamethasone recentor binding in isol ated cytosols. Isolated HeLa S3 cell; or thymocytes were incubated with loM [SHldexamethasone for 2 h at 7 C. Aliquots (0.9 ml) of cytosol were brought to 5 NM 5'-deoxynyridoxal with the addition of 0.1 ml of 50 ml4 stock solution (PH 7.6). Controls received an equivalent amount of 1.5 mM MgC12., At intervals thereafter 100 ~1 aliquots of cytosol were removed, treated with 100 ~1 of dextran-coated charcoal for 5 min and centrifuged at 10,000 x g for 1 min. Samples of the supernates were placed into scintillation vials for the determination of radioactivity. The data was nlotted as the % of the control [SH]dexamethasone binding observed in 5'deoxyoyridoxal-treated cytosol. [3H]Dexamethasone binding in control cytosols is indicated by open circles. Data from three separate exneriments are plotted
only
represented
thymus
5-10%
cytosols.
show
that
whole
These
at at
Several
at 2°C. little
decrease
similar
loss
to
control
those
(37°C)
and
next
examined
HeLa
S3 and
represented
glucocorticoid
the of
were
thymocyte
rat
concentration
in
importance by
decrease
analogs
in
in
Fig
receptor
binding
isolated
cytoplasmic
of
on
the
In
cytosol
the
5 mM were
of failure steroid-receptor
the of
of incubated
of
added
1, in
effect
receptors
to
thymus
bound
with
alcohol In
159
cytosol
for
5'-deoxypyridoxain
5 mM 5'-deoxypyridoxal,
group
addition,
was of
a
observed
after
5'-deoxypyridoxal or
amine the
3).
5'-deoxy-
[3tt]dexamethasone
[3H]dexamethasone
corresponding binding.
bound
on (Fig
or
and
saturable
C4'-carboxaldehyde the
their
cytoplasmic
5'-deoxypyridoxine,
amount
amount
for
5'-deoxypyridoxamine
5'-phosphate,
effect
six-fold
indicated
in
with
temperatures
Pyridoxal
preparations.
The
binding
conjunction
5'-deoxypyridoxine,
a final
cytosol
2 h.
can
binding
pyridoxal
had
in
5'-deoxypyridoxal
5'-phosphate,
mine
data,
initial
2°C.
Pyridoxal
at
total
physiological
[3H]dexamethasone
2 hr
the
5'-deoxypyridoxal
cells
fractions
of
to
presence
is effect
a of
the
Vol. 92, No. 1, 1980
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
too I-
Figure if3. The specificity of 5'-deoxypyridoxal action on cytoplasmic dexamethason receptor. Thymocytes were incubated at 2OC for 2 h with either 2 x 10m8 M [ 5 Hldexanethasone alone or [3Hldexamethasone plus 2 x 10e6 M unlabeled dexamethasone. Cytosols were prepared and incubated with 5 mM of the indicated compounds at 2OC for an additional 2 h. Receptor binding was measured with the dextran-coated charcoal assay and saturable binding values (mean i S.E., 2 determinations) are plotted.
apolar
methyl
substituent
glucocorticoid charged
at
receptor C5'
phosphate
receptor
binding.
specific
manner
appears
These
to
is
essential
for
5'-phosphate
ineffective
data.indicate
reduce
be
Pyridoxal
binding.
group,
to
C5'
that
glucocorticoid
with
in causing
a loss
5'-deoxypyridoxal receptor
the
reduction
a highly
of steroid acts
binding
in
in
in
these
a highly
prepara-
tions.
Discussion We have Bg analog, receptor
demonstrated decreases
complexes
in
isolated
cytoplasmic
of
[3H]dexamethasone
that the
in
5'-deoxypyridoxal,
binding
of
S3 cells
HeLa
fractions binding
dexamethasone and at
was
a potent
found
rat 2°C. for
160
to
synthetic
nuclear
thymocytes
--in
glucocorticoid vitro
A rapid
and
nuclear
receptor
vitamin
nearly
at
37°C
and
complete complexes
loss in
whole
Vol.
92. No.
HeLa
BIOCHEMICAL
1. 1980
S3 cells
and
rat
5'-deoxypyridoxal, mocyte
with
cytosol
be quite as
from than
at
specific
for
in
2"C,
5'-phosphate,
RESEARCH
37°C
cell
in
the
viability.
similar
dissociation
were
control
producing
at in
complexes
found
BIOPHYSICAL
incubated
alteration
receptor
those
pyridoxal
no
preparations
dexamethasone greater
thymocytes
AND
presence
of
1 mM
In
S,
and
HeLa
rates
calculated,
cytosols.
COMMUNICATIONS
of
approximately
in
5'-deoxypyridoxal
appears
glucocorticoid and
[3H]-
20-fold
5'-Deoxypyridoxal
reductions
bound
thy-
receptor
to binding,
5'-deoxypyridoxamine
were
ineffective. 5'-Deoxypyridoxal decrease
the
in
cells.
whole
sult
of
its
binding
of
with its
the
be
a non-steroidal
steroid
B6 receptor
as
to
effects
vitamin
potential
to
glucocorticoids
Whether inherent
interaction theless,
appears
of
antagonist remains
a non-steroidal
compound
specific
receptor
5'-deoxyoyridoxal action to
be
due
established.
glucocorticoid
can
proteins are
or
which
to
the
re-
a direct Never-
antagonist
is
suggested.
Acknowledgements -_-Supported
in
part
by
NIH
grants
AM 20892
and
AM 20762.
References ___1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13.
Cidlowski, J.A. and Thanassi, J.W. (1978) Biochem. Biophys. Res. Commun. 82, 1140-1146. Cidlowski, J.A. and Thanassi, J.W. (1979) Biochemistry 18, 2378-2384. Cake, M.H., DiSorbo, D.M. and Litwack, G. (1978) J. Biol. Chem. 253, 4886-4891. Mainwaring, W.I.P. (1975) in Vitamins and Hormones (Munson, P.L., Glover, J Diczfalusy, E. and Olsen, R.E., eds) Vo1.33, PP. 223-245, Academic Pi&s, New York. Carbone, P.P. (1975) Ann. Intern. Med. 83, 730-731. Neri, R. and Monahan, M. (1972) Invest. Urol. 10, 123-130. Marver, D., Stewart, J., Funder, J., Feldman, D., and Edelman, I. S. (1974) Proc. Natl. Acad. Sci. 71, 1431-1435. Terenius, L. (1971) Eur. J. Cancer 7, 57-64. Horwitz, K.B. and McGuire, W.L. (1978) J. Biol. Chem. 253, 8185-8191. Korennan, S.G. (1968) Endocrinology 87, 1119-1123. Munck, A. and Wira, C. (1971) in Advances in the Biosciences (Raspe, G. ed.) Vol 7, Perganon Press, Vieurg pp. 301-330. Kaiser, N., Milholland, R., Turnell, R.W. and Rosen, F. (1972) Biochem. Biophys. Res. Commun. 49, 516-521. Turnell, R.W., Kaiser, N., Milholland, R.J. and Rosen F. (1974) 249, 1133-1138.
161
Vol.
92, No.
14.
;65: K:
19.
1, 1980
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Melnykovych, G. and Bishop, C.F. (1969) Biochem. Biouhys. Acta. 177, 579-585. Munck, A. (1968) J. Biol. Chem. 243, 1039-1042. Mijhlradt, D.F. and Snell, E.E. (1967) J. Med. Chem. 10, 129-130. Cunningham, W.C., and Thanassi, J.W. (1979) Experientia 35, 451-452. Munck, A. and Wira, C. (1975) in Methods in Enzymology (O'Malley, B.W. and Hardman, J., eds.) Vol. 36, Academic Press, New York, pp. 255-266. Sibley, C.H. and Tomkins, G.M. (1974) Cell 2, 221-227.
162