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Succinylacetone, a potent inhibitor of heme biosynthesis: Effect on cell growth, heme content and δ-aminolevulinic acid dehydratase activity of malignant murine erythroleukemia cells
Vol. 88, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
June 27, 1979
Pages 1382-1390
SUCCINYLACETONE, A POTENT INHIBITOR OF HEME BIOSYNTHESIS: EFFECT ON CELL GROWTH, HEME CONTENT AND 6-AMINOLEVULINIC ACID DEHYDRATASE ACTIVITY OF MALIGNANT MURINE ERYTHROLEUKEMIA CELLS Paul S. Ebert Laboratory of Molecular Virology National Cancer Institute, N. I. H. Bethesda, Maryland 20205 Richard A. Hess, Bruce C. Frykholm, and Donald Metabolism Branch, National Cancer Institute, Bethesda, Maryland 20205
Received
May
16,
P. Tschudy N.I.H.
1979 SUMMARY
4,6-Oioxoheptanoic acid (succinylacetone, SA) was examined with regard to its ability to a) inhibit the second enzyme of the heme pathway, b-aminolevulinic acid (ALA) dehydratase, b) lower the heme concentration, and c) inhibit SA profoundly cell growth of murine erythroleukemia (MEL) cells in culture. inhibited ALA dehydratase in broken cell preparations at concentrations as low as lo-' M. The stimulation of hemoglobin production by DMSO and butyrate in MEL cells was inhibited by the addition of SA to the cell medium. When 1 mM SA was added to the medium, there was a profound inhibition of ALA dehydratase activity, and the heme concentration of cells declined progressively with each cell division. Cell growth was markedly inhibited after two cell divisions.
INTRODUCTION It excrete
has recently
been shown that
a metabolite
which
dehydratase
(1).
is derived
from
excretion which
have previously chelating
non-specific
is an inhibitor
compound,
tyrosine
observed
triazole,
and is
in these
agents,
of ALA dehydratase
more specific
thought
patients
available
@ 1979
by Academic Press, Inc. in any form reserved.
of reproduction
acid
to be the cause (1).
Most
tyrosinemia
weak inhibitors
phosphate
as an inhibitor
of the
SA)
increased
ALA
of heme biosynthesis
levulinate,
3-amino-1,2,4-
inhibitors,
are either
later,
The very potent suggests
that
it
is
of heme biosynthesis
The present
1382
(ALA)
(succinylacetone,
of the pathway.
by SA, as presented
inhibitors.
acid
inhibitors
such as lead,
0006-291X/79/121382-09$01.00/0 Copyright All rights
hereditary
of &aminolevulinic
and pyridoxal
and much more active
the previously
with
4,6-dioxoheptanoic
been available,
or relatively
inhibition
than
This
patients
study
was undertaken
to
BIOCHEMICAL
Vol. 88, No. 4, 1979
determine
the effects
heme concentration
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of inhibition and growth
of ALA dehydratase
of murine
erythroleukemia
MATERIALS 4,6-Dioxoheptanoic reagents
were
method
of Gibson
of reagent
In the studies was measured
al -et --
with
added
washing
was omitted All
aerobically. bovine with
with
for
as a standard.
ALA to form a pyrrole
mM ALA (pH 7.0), was measured
to the boiled
nm in a Model calculated
using
starting line
procedure cells frozen
addition mixture.
a molar
concentrations
pyrrole
derivative. of Sassa --et al.
were
washed
prior
of the method
for
by the method
twice
of an equal
volume
The resultant
pink
extinction
activity
minor
as described
by the oxalic
Hemoglobin (6)
as described
1383
after
reaction
The reaction 1 ml of 10 The pyrrole reagent
was read
which than
was based
by Ebert
on
the crystal-
acid-fluorometric cells.
in 0.5 ml water,
was measured
at 556
of SA was
on 4~10~
suspended
performed using
Ehrlich's
rather
modifications,
60 min at
(3)
(pH 4.6).
complex
solutions,
above,
activa-
were
condensation.
color
experi-
Sulfhydryl
of 3.8~10~
Heme was determined
other
freeze-thawed
The concentration
of SA in standard
and Furth
were
of modified
coefficient
to heme determination. of Crosby
In all
1.5 mM ALA for
buffer
1 mM
to
of 1 ml of medium,
acetate
l),
had been neutralized
of Lowry --et al.
20 min a mixture
with
(SA)
SA in medium was measured
Spectrophotometer.
(5)
(Table
NaCl.
with
enzyme activity
added
of ALA dehydratase
and 1 ml of 1 M sodium
240 Gilford
7.4)-0.9%
by the
were
which
by means of a Knorr-type
by boiling
after
cells
incubated
measurement
serum albumin
preparations
60 min at 37'.
on 4~10~
were
was measured
out
for
1 mM tris(pH
lysates
in which
which
other
of experiments.
of inhibitor
concentration)
Protein
was carried
(4)
concentrations
was continued
incubations
cell
All
was measured
of ALA dehydratase
was measured
on the
cells.
Inc.
in two types
indicated
and the
Proteochem,
modifications
in broken
twice
(MEL)
ALA dehydratase
inhibitor
The incubation
once after
37O.
with
to 5 mM ALA (final
ments ALA dehydratase
from
quality.
inhibition
and the
10 min prior
tion
(2)
compound
AND METHODS
was obtained
grade
of direct
dithiothreitol
pH 6.8.
acid
by this
The and
by a modification --et al.
(7).
Vol. 88, No. 4, 1979
MEL cells
8lOCHEMlCALAND
from
clone
745 were
routinely
containing
heat-inactivated
glutamine,
and penici11in:streptomycin:neomycin
previously
(7).
2.
These
conditioned
fetal
bovine
conditions
In the experiments
were
reported
to low protein
experiments
serum.
For the experiments conditioned
(modified) neomycin Viable
reported
cells
medium containing (as above), cells
were
in Figure
cells
were
pre-
2, in which
centrifuged,
and resuspended
2.5% gelatin
(DIFCO),
for
bovine
1, the medium contained
serum.
1% fetal
defined
2
bovine
medium was
in Ham's
F-12
penicillin:streptomycin:
and 30 nM insulin
in a hemacytometer
in Table
the MEL cells fetal
2 mM
as described
reported
1 and 2, stock
in Figure
lo%),
pg/ml)
used in experiments
2% heat-inactivated
2 mM glutamine,
monitored
concentration
by maintaining
reported
were
in RPM1 1640 medium
(10:10:20
concentrations
For the
maintained serum (final
in Figures
days in RPM1 1640 medium containing
used,
BIOPHYSICAL RESEARCH COMMUNICATIONS
by the
(Sigma trypan
Chemical blue
Co.).
exclusion
method. TABLE
1.
Inhibition
of
ALA dehydratase erythroleukemia
by succinylacetone cells.
(SA)
Enzyme activity (nmoles PBG/mg protein/hr)
murine
Non-induced
Induced SA Concentration W
in
% Inhibition
Enzyme activity (nmoles PBG/mg protein/hr)
% Inhibition
0
35.1
0.0
8.8
0.0
10-8
26.7
23.9
6.8
22.7
10-7
3.8
89.2
1.1
87.5
10-6
0.9
97.4
0.2
97.7
10-5
0.3
99.1
0.1
98.9
10-4
0.0
0.0
100
100
MEL cells were grown in RPM1 1640 containing 1 or 10% fetal bovine serum for three days. The data under the column heading "Induced" are from cells in which hemoglobin synthesis was induced by addition of 1 mM butyrate to medium containing 10% fetal bovine serum on day zero. The data under the column heading "Non-induced" are from cells grown in medium containing I.% fetal bovine serum. ALA dehydratase activity was measured in the presence of the indicated concentrations of SA. Cells were washed twice in normal saline and the pellet was freeze-thawed five times after which it was resuspended in 0.14 M KCl. The incubation mixture consisted of 1 ml of cell suspension, 1 ml of 0.07 M potassium phosphate buffer (pH 6.8), 1 ml of 3 mM dithiothreitol, and 30 pl of SA solution to give the final concentrations indicated. After 10 min of incubation in a Oubnoff incubator at 37' with shaking, 0.3 ml of 50 mM ALA (neutralized to pH 6.8) was added. The incubation was continued for 60 min, and was ended by the addition of 1 ml of dialyzed iron and 2 drops of saturated copper sulfate. The porphobilinogen (PBG) was determined after mixing equal volumes of supernatant solution and modified Ehrlich's reagent.
1384
Vol.
88,
No.
BIOCHEMICAL
4, 1979
TABLE
2.
Effect
AND
of succinylacetone stimulated by inducers
BIOPHYSICAL
on hemoglobin of hemoglobin
COMMUNICATIONS
concentration synthesis
of
pg Hemoglobin/ lo6 cells
Cells x106/ml
Treatment
RESEARCH
of
MEL cells
% decrease hemoglobin
None
4.6
0.2
2% OMSO
4.8
1.8
0
2% DMSO + 0.01 mM SA
4.7
1.3
28
2%DMSO+
O.lmMSA
5.2
0.9
50
2% DMSO +
0.6
5.6
0.4
78
4.4
0.9
0
3.8
0.4
56
mM SA
1 mM butyrate 1 mM butyrate
+ 0.1
mM SA
--
MEL cells, subdivided to a concentration of 105/ml, were incubated in RPM1 1640 medium containing 10% fetal bovine serum with either inducer or inducer and SA for 5 days. Cells were counted and total cell growth is reported under Cells were then centrifuged and washed. the column heading "Cellsx106/ml.' Aliquots of 2~10~ total cells were assayed for hemoglobin concentration (7).
RESULTS
The MEL
effect
cells
was
stimulated
by
low
the
as
lo-*
inhibition
both
and
sively
depressed
ments
is
growth
and the
2.
cells
to
SA at about
Table to
low
effect
the
of
the
at
at
SA to
of
hemoglobin of
of as
As
concentration
which
had
inhibition
is
seen
in
been
of
at
expected,
ALA
concentrations the
magnitude
concentration
in
cultures
seen
DMSO,
of
1385
SA.
In
mM) the
of
SA for
in cells.
cells
in
the
dimethylsulfoxide content
was
a variety
of
varying
DMSO
synthesis
DMSO-treated
MEL
hemoglobin of
with
of
synthesis,
SA (0.1-0.5
hemoglobin as
cells
a given
concentrations
mM inhibited
in
heme
profound
7 M.
adding
presence
observed,
extent
lo-
on
enzyme.
concentrations
same
and
Inhibition
same
inducers
increasing
were 0.1
90%
and
The
1.
non-induced
In by
cells
synthesis.
the
different
stimulation
Table
in
close
the
activity
untreated
approximately
butyrate.
involving
heme
M and
two
(DMSO)
of shown
2 shows of
dehydratase
in
SA is
induced
Table
ALA
both
inducers
is
the
presence
SA on
studied
by
dehydratase as
of
degrees
experiment butyrate-treated
progresexperiof
reported
in
Vol. 88, No. 4, 1979
BIOCHEMICAL
I 1
L 0
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
I
I
I
I
I
2
3
4
5
6
7
DAYS
FIGURE 1. Heme concentration in MEL cells induced with butyrate with and without SA. MEL cells were grown in RPM1 1640 containing 1% fetal bovine serum. 1 mM butyrate and 0.1 mM SA were present for the entire 7 day heme concentration in cells grown in medium incubation period. o-o: containing 1 mM butyrate. o-o: heme concentration in cells grown in medium containing 1 mM butyrate and 0.1 mM SA.
The heme content with
those
grown
a decreased
Though
inhibition than
fetal
bovine
entered cells
and that
(Table
2),
taken
cells
Fetal
it
Figure
occurred,
uninduced
the the
MEL cells
(Figure
appeared
entire
compared
1.
The SA
7 day period.
levels
observed
grown
in medium
bovine
ALA dehydratase
to inhibit
2).
defined
Furthermore, bovine
because
2 shows the points
kinetics in the
1% fetal unknown
serum were
were
never
containing
1386
l),
that
synthesis
in induced
in Figure
2, the as the protein
was shown to contain bovine
serum)
it
was investigated
gelatin
which
heme might
be
of growth-promoting
by the
growth
curve
it
effects
obviated
of cell growth
shown
medium containing
serum was avoided
(Table
and growth
In the experiments
of medium containing
in fetal
hemoglobin
on heme concentration
in completely
heme/ml
at various
SA inhibited
effect
up by the cells.
substances
cells
its
grown
(79 pmoles
for
was shown that
cells,
source.
observed
seen in Figure
throughout
of heme synthesis
it
were
+ 0.1 mM SA is
of 1 mM butyrate
serum.
in uninduced cells
in the presence
heme concentration
those
After
grown
in 1 mM butyrate
caused
lower
of cells
use of gelatin.
and the
in response
heme concentrations to the continuous
of
Vol.
88,
No.
BIOCHEMICAL
4, 1979
AND
BIOPHYSICAL
RESEARCH
A
4r
COMMUNICATIONS
El
A
j
70
3 -/
2 F
70 c
3
60
i
I
:;4!i 0
FIGURE
3
4
5 DAYS
6
7
8
9
10
2. Growth characteristics and heme concentration in MEL cells grown defined medium with and without SA. MEL cells were grown in modified F-12 medium supplemented with 2.5% gelatin and 30 nM insulin. On days and 7 the cells were subdivided to concentrations of 3~10~ live cells/ml in fresh medium with or without SA as indicated. Growth curves beyond day 3 are corrected to show continuous growth. o-o: cell growth. O-0: heme concentration. Panel A shows the growth pattern and heme concentration of MEL cells in medium without SA. Panel B shows the growth properties and heme concentration of cells grown in the above Panel C shows growth kinetics and heme medium containing 0.1 mM SA. concentration of cells grown in the above medium containing 1 mM SA.
exposure until
of day
increased below
12
50
44
MEL
to
7 at
which
time
cell
counts
until
day
3 and
then
gradually
pmoles
heme/mg
SA.
Control
cells
protein.
In
cells
(panel
began
to
A)
decrease.
decreased. contrast
1387
continued
to
Heme the
untreated
to
divide
Heme
concentrations
levels
never cells,
dropped MEL
in 3
Vol.
88,
No.
cells
4, 1979
exposed
to
experimental
0.1
cells levels
control
in
mM SA (Panel
cell
growth
day
cells.
the
rate
protein,
heme
cell
death
the
increased
cellular l/6
heme
the
lowest
concentrations
the
entire
day
heme/mg
0.1 When
COMMUNICATIONS
for third
pmoles
containing
initially
about
38
sustained.
point
divide
until to
medium
RESEARCH
to
increased
was
which
Cellular of
in
BIOPHYSICAL
continued
decreased
growth
3 at
heme/mg
MEL
cell
B)
levels
slowly grown
C),
on
pmoles
then
cells
levels,
density
Heme
and
AND
mM SA (panel
period.
control heme
8lOCHEMlCAL
as
with
Though
slightly
MEL
cells
were
and
cells
reached
concentrations
below
exposed
decline
1
maximum to
observed
to
to
dropped
concentration
day
the
protein.
mM SA were
continued
10
in
7 untreated
thereafter,
and
increased.
DISCUSSION
The in
MEL
present cells
presented sible
rat
liver
of
the
of
dehydratase
The
effect
in
which
butyrate. by
In
the
lowered With
the
heme
reaches
the
inhibitor of ALA rate
alone
an
increased
synthatase. of
of
heme
at
of
of
to
significantly
induced
SA appeared
to
diminish
the
cells
of
inhibitor
cells
grown
in
3 times
those
seen
of least (Figure
2). size
of
This
increased
biosynthesis
in
reason
for
ALA
caused
by
the
this the
cells
medium
grown is
MEL
such
of
thought
as
even
turn if
as
it
in
the to
and
2). seen
butyrate
and
evidenced
Table is
that
(Figure
1)
presence
of
be
the
result
induction leads
the
cells
DMSO
butyrate-mediated in
great
production.
1 & 2,
the
irrever-
inhibition
synthesis,
presence
cells
1388
agents
be
in
untreated
heme
concentration
induced
heme
be
unpublished
profound
by
in
to
present
(Figures
the
The
ALA
is
both
in
it B.C.,
diminish
was
dehydratase will
demonstrate
in
ALA
inhibition
biosynthesis,
synthesis
concentration
the
tissues
examined
of
of
Frykholm,
most
heme
was
will
R.A.,
in
concentration
pool
kinetics
Hess,
compound
cases,
same
the
enzyme needed
this
heme
inhibition
dehydratase
hemoglobin both
profound
which
D.P.,
rate-limiting is
of
the
publication,
ALA
concentration levels
Details
(Tschudy, Since
excess
those
1).
a subsequent
observations).
ALA
demonstrates
(Table in
in
study
amount
to
a greater of
active
of
Vol.
88,
No.
ALA
dehydratase
model
of
4, 1979
is
this
It
BIOCHEMICAL
is
the
type seen
of
heme
concentration
which
would
approach
tions
(Table
1). does
inhibit
ALA
the
period.
In
heme
the was
shown
MEL
heme
in
experiment
be
present
of In
fetal these
the
bovine
below
heme
biosynthesis,
a)
intracellular
in
which
presumed when in
Figure
concentration
of
inhibition
in
a completely (Figure
10
to
pmoles
should
with
heme the
2 suggest decreases
that
effect cell to
that
by
of
heme
of
the
than
a 3 day was
fact
that at
those
the
possibility
cells,
the
a in that
final
biosynthesis
by
using
by
the
serum
higher
medium
up
over
measurement
MEL
profoundly
taken
one
of
up
In
heme
SA on
gelatin
b)
decrease degradation
in
dilution is
l/6
1389
can
these
on decline from about
cells
heme
by
would cell
lower
to
inhibition
when level
of
of
by
2 mechanisms:
cell
division
50%. be
1
progressive
It
a minor
division.
accelerated the
of
declined
resulting
rapidly
approximately
with
concentration
of
presence
experiments
dilution
in
the
concentrations
these
heme
in
concentration
concentrations
or
death
grown
heme heme
protein.
of
view
Because
cells
in when
degradation,
division
the
but
bovine
in was
defined
shown
intracellular
Either
2).
rapidly
heme/mg
prepara-
measured,
in
that
to be
markedly
cell
constant
serum
taken
decrease
die
not
possible,
1 & 2).
effect
broken
may
fetal
which
be
a pronounced
intracellular
compared
Tables
medium
which
protein)
was
the
bovine
might
it
heme
each
fetal
serum
serum
began
is
than
considered.
approximately
to
cells
heme/mg
experiments
Cells
levels
the
1,
in
SA were
be
medium
in
conducted
mM SA experienced growth.
by
(Figure bovine
was
to
higher
to
concentration
in of
necessary
much
be
A quantitative
(8,9).
dehydratase must
heme
appeared
pmoles
involving
growth
place
heme
cells
fetal
of
involving
of
is
COMMUNICATIONS
cells.
medium
intracellular
traces
medium
the 2)
explanations
or
uninduced
SA in
ALA
concentrations
(269
untreated
cell
to
of
RESEARCH
discussed
(Figure
sufficient
studies
uptake
concentration
the
achieve
the
the
of
cells
possible
and
previously
inhibition
Intracellular in
been
the
BIOPHYSICAL
induced
concentration
100% Two
both has
in
not
concentration
added,
the
dehydratase,
cells.
in
system
that
lower
inhibitor
same
AND
factor The
the
heme
untreated
is
data
Vol. 88, No. 4, 1979
cells.
With
this
level
cell
divisions
a 50% dilution
would
a) diminished decreased of protein cells
for
be reached observed
The killing doublings
BIOCHEMICAL
effect
and normal
division
2 cell
divisions. death
of SA on MEL cells
which
of 1 mM SA could
result
respiration
resulting
synthesis.
synthesis
each cell
when accelerated
in the presence
protein
after
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in several
from
lowered
it
would This
be expected
was the number
of cells
of
occurred. had progressed
through
from 2 possible cytochrome
levels,
Heme has been shown to be involved types
that
(10,11,12),
including
2
mechanisms: and/or
b)
in initiation both
MEL
reticulocytes.
REFERENCES 1.
2. 3. Z:
F: 8. 9. 10. 11. 12.
Lindblad, B., Lindstedt, S., Steen, G. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4641-4645. Gibson, K. Cl., Neuberger, A., Scott, J. J. (1956) Biochem. J. (London) 61, 618-629. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. (1957) J. Biol. Chem. 193, 265-275. Mauzerall, D., Granick, S. (1956) J. Biol. Chem. 219, 435-446. Sassa, S., Granick, S., Chang, C., Kappas, A. (1974) In Erythropoiesis, Proc. of the 4th Int. Conf. on Erythropoiesis. Eds. Nakao, K., Fischer, J. W., Takaku, F., pp. 383-396. Crosby, W. H. and Furth, F. W. (1956) Blood 11, 380-383. Ebert, P. S., Wars, I. M. Buell, D. N. (1976) Cancer Res. 36, 1809-1813. Tschudy, D. P., Bonkowsky, H. L. (1973) Molec. Cell. Biochem. 2, 55-62. Tschudy, D. P. (1973) Molec. Cell Biochem. 2, 63-70. Dabney, 8. J. and Beaudet, A. L. (1978) J. Biol. Chem. 253, 7124-7126. Datta, A., DeHaro, C., Sierra, J. M., Ochoa, S. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3326-3329. London, I. M., Clemens, M. J., Ranu, R. S., Levin, D. H., Cherbas, L. F., and Ernst, V. (1976) Fed. Proc. 35, 2218-2222.
1390
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
June 27, 1979
Pages 1382-1390
SUCCINYLACETONE, A POTENT INHIBITOR OF HEME BIOSYNTHESIS: EFFECT ON CELL GROWTH, HEME CONTENT AND 6-AMINOLEVULINIC ACID DEHYDRATASE ACTIVITY OF MALIGNANT MURINE ERYTHROLEUKEMIA CELLS Paul S. Ebert Laboratory of Molecular Virology National Cancer Institute, N. I. H. Bethesda, Maryland 20205 Richard A. Hess, Bruce C. Frykholm, and Donald Metabolism Branch, National Cancer Institute, Bethesda, Maryland 20205
Received
May
16,
P. Tschudy N.I.H.
1979 SUMMARY
4,6-Oioxoheptanoic acid (succinylacetone, SA) was examined with regard to its ability to a) inhibit the second enzyme of the heme pathway, b-aminolevulinic acid (ALA) dehydratase, b) lower the heme concentration, and c) inhibit SA profoundly cell growth of murine erythroleukemia (MEL) cells in culture. inhibited ALA dehydratase in broken cell preparations at concentrations as low as lo-' M. The stimulation of hemoglobin production by DMSO and butyrate in MEL cells was inhibited by the addition of SA to the cell medium. When 1 mM SA was added to the medium, there was a profound inhibition of ALA dehydratase activity, and the heme concentration of cells declined progressively with each cell division. Cell growth was markedly inhibited after two cell divisions.
INTRODUCTION It excrete
has recently
been shown that
a metabolite
which
dehydratase
(1).
is derived
from
excretion which
have previously chelating
non-specific
is an inhibitor
compound,
tyrosine
observed
triazole,
and is
in these
agents,
of ALA dehydratase
more specific
thought
patients
available
@ 1979
by Academic Press, Inc. in any form reserved.
of reproduction
acid
to be the cause (1).
Most
tyrosinemia
weak inhibitors
phosphate
as an inhibitor
of the
SA)
increased
ALA
of heme biosynthesis
levulinate,
3-amino-1,2,4-
inhibitors,
are either
later,
The very potent suggests
that
it
is
of heme biosynthesis
The present
1382
(ALA)
(succinylacetone,
of the pathway.
by SA, as presented
inhibitors.
acid
inhibitors
such as lead,
0006-291X/79/121382-09$01.00/0 Copyright All rights
hereditary
of &aminolevulinic
and pyridoxal
and much more active
the previously
with
4,6-dioxoheptanoic
been available,
or relatively
inhibition
than
This
patients
study
was undertaken
to
BIOCHEMICAL
Vol. 88, No. 4, 1979
determine
the effects
heme concentration
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of inhibition and growth
of ALA dehydratase
of murine
erythroleukemia
MATERIALS 4,6-Dioxoheptanoic reagents
were
method
of Gibson
of reagent
In the studies was measured
al -et --
with
added
washing
was omitted All
aerobically. bovine with
with
for
as a standard.
ALA to form a pyrrole
mM ALA (pH 7.0), was measured
to the boiled
nm in a Model calculated
using
starting line
procedure cells frozen
addition mixture.
a molar
concentrations
pyrrole
derivative. of Sassa --et al.
were
washed
prior
of the method
for
by the method
twice
of an equal
volume
The resultant
pink
extinction
activity
minor
as described
by the oxalic
Hemoglobin (6)
as described
1383
after
reaction
The reaction 1 ml of 10 The pyrrole reagent
was read
which than
was based
by Ebert
on
the crystal-
acid-fluorometric cells.
in 0.5 ml water,
was measured
at 556
of SA was
on 4~10~
suspended
performed using
Ehrlich's
rather
modifications,
60 min at
(3)
(pH 4.6).
complex
solutions,
above,
activa-
were
condensation.
color
experi-
Sulfhydryl
of 3.8~10~
Heme was determined
other
freeze-thawed
The concentration
of SA in standard
and Furth
were
of modified
coefficient
to heme determination. of Crosby
In all
1.5 mM ALA for
buffer
1 mM
to
of 1 ml of medium,
acetate
l),
had been neutralized
of Lowry --et al.
20 min a mixture
with
(SA)
SA in medium was measured
Spectrophotometer.
(5)
(Table
NaCl.
with
enzyme activity
added
of ALA dehydratase
and 1 ml of 1 M sodium
240 Gilford
7.4)-0.9%
by the
were
which
by means of a Knorr-type
by boiling
after
cells
incubated
measurement
serum albumin
preparations
60 min at 37'.
on 4~10~
were
was measured
out
for
1 mM tris(pH
lysates
in which
which
other
of experiments.
of inhibitor
concentration)
Protein
was carried
(4)
concentrations
was continued
incubations
cell
All
was measured
of ALA dehydratase
was measured
on the
cells.
Inc.
in two types
indicated
and the
Proteochem,
modifications
in broken
twice
(MEL)
ALA dehydratase
inhibitor
The incubation
once after
37O.
with
to 5 mM ALA (final
ments ALA dehydratase
from
quality.
inhibition
and the
10 min prior
tion
(2)
compound
AND METHODS
was obtained
grade
of direct
dithiothreitol
pH 6.8.
acid
by this
The and
by a modification --et al.
(7).
Vol. 88, No. 4, 1979
MEL cells
8lOCHEMlCALAND
from
clone
745 were
routinely
containing
heat-inactivated
glutamine,
and penici11in:streptomycin:neomycin
previously
(7).
2.
These
conditioned
fetal
bovine
conditions
In the experiments
were
reported
to low protein
experiments
serum.
For the experiments conditioned
(modified) neomycin Viable
reported
cells
medium containing (as above), cells
were
in Figure
cells
were
pre-
2, in which
centrifuged,
and resuspended
2.5% gelatin
(DIFCO),
for
bovine
1, the medium contained
serum.
1% fetal
defined
2
bovine
medium was
in Ham's
F-12
penicillin:streptomycin:
and 30 nM insulin
in a hemacytometer
in Table
the MEL cells fetal
2 mM
as described
reported
1 and 2, stock
in Figure
lo%),
pg/ml)
used in experiments
2% heat-inactivated
2 mM glutamine,
monitored
concentration
by maintaining
reported
were
in RPM1 1640 medium
(10:10:20
concentrations
For the
maintained serum (final
in Figures
days in RPM1 1640 medium containing
used,
BIOPHYSICAL RESEARCH COMMUNICATIONS
by the
(Sigma trypan
Chemical blue
Co.).
exclusion
method. TABLE
1.
Inhibition
of
ALA dehydratase erythroleukemia
by succinylacetone cells.
(SA)
Enzyme activity (nmoles PBG/mg protein/hr)
murine
Non-induced
Induced SA Concentration W
in
% Inhibition
Enzyme activity (nmoles PBG/mg protein/hr)
% Inhibition
0
35.1
0.0
8.8
0.0
10-8
26.7
23.9
6.8
22.7
10-7
3.8
89.2
1.1
87.5
10-6
0.9
97.4
0.2
97.7
10-5
0.3
99.1
0.1
98.9
10-4
0.0
0.0
100
100
MEL cells were grown in RPM1 1640 containing 1 or 10% fetal bovine serum for three days. The data under the column heading "Induced" are from cells in which hemoglobin synthesis was induced by addition of 1 mM butyrate to medium containing 10% fetal bovine serum on day zero. The data under the column heading "Non-induced" are from cells grown in medium containing I.% fetal bovine serum. ALA dehydratase activity was measured in the presence of the indicated concentrations of SA. Cells were washed twice in normal saline and the pellet was freeze-thawed five times after which it was resuspended in 0.14 M KCl. The incubation mixture consisted of 1 ml of cell suspension, 1 ml of 0.07 M potassium phosphate buffer (pH 6.8), 1 ml of 3 mM dithiothreitol, and 30 pl of SA solution to give the final concentrations indicated. After 10 min of incubation in a Oubnoff incubator at 37' with shaking, 0.3 ml of 50 mM ALA (neutralized to pH 6.8) was added. The incubation was continued for 60 min, and was ended by the addition of 1 ml of dialyzed iron and 2 drops of saturated copper sulfate. The porphobilinogen (PBG) was determined after mixing equal volumes of supernatant solution and modified Ehrlich's reagent.
1384
Vol.
88,
No.
BIOCHEMICAL
4, 1979
TABLE
2.
Effect
AND
of succinylacetone stimulated by inducers
BIOPHYSICAL
on hemoglobin of hemoglobin
COMMUNICATIONS
concentration synthesis
of
pg Hemoglobin/ lo6 cells
Cells x106/ml
Treatment
RESEARCH
of
MEL cells
% decrease hemoglobin
None
4.6
0.2
2% OMSO
4.8
1.8
0
2% DMSO + 0.01 mM SA
4.7
1.3
28
2%DMSO+
O.lmMSA
5.2
0.9
50
2% DMSO +
0.6
5.6
0.4
78
4.4
0.9
0
3.8
0.4
56
mM SA
1 mM butyrate 1 mM butyrate
+ 0.1
mM SA
--
MEL cells, subdivided to a concentration of 105/ml, were incubated in RPM1 1640 medium containing 10% fetal bovine serum with either inducer or inducer and SA for 5 days. Cells were counted and total cell growth is reported under Cells were then centrifuged and washed. the column heading "Cellsx106/ml.' Aliquots of 2~10~ total cells were assayed for hemoglobin concentration (7).
RESULTS
The MEL
effect
cells
was
stimulated
by
low
the
as
lo-*
inhibition
both
and
sively
depressed
ments
is
growth
and the
2.
cells
to
SA at about
Table to
low
effect
the
of
the
at
at
SA to
of
hemoglobin of
of as
As
concentration
which
had
inhibition
is
seen
in
been
of
at
expected,
ALA
concentrations the
magnitude
concentration
in
cultures
seen
DMSO,
of
1385
SA.
In
mM) the
of
SA for
in cells.
cells
in
the
dimethylsulfoxide content
was
a variety
of
varying
DMSO
synthesis
DMSO-treated
MEL
hemoglobin of
with
of
synthesis,
SA (0.1-0.5
hemoglobin as
cells
a given
concentrations
mM inhibited
in
heme
profound
7 M.
adding
presence
observed,
extent
lo-
on
enzyme.
concentrations
same
and
Inhibition
same
inducers
increasing
were 0.1
90%
and
The
1.
non-induced
In by
cells
synthesis.
the
different
stimulation
Table
in
close
the
activity
untreated
approximately
butyrate.
involving
heme
M and
two
(DMSO)
of shown
2 shows of
dehydratase
in
SA is
induced
Table
ALA
both
inducers
is
the
presence
SA on
studied
by
dehydratase as
of
degrees
experiment butyrate-treated
progresexperiof
reported
in
Vol. 88, No. 4, 1979
BIOCHEMICAL
I 1
L 0
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
I
I
I
I
I
2
3
4
5
6
7
DAYS
FIGURE 1. Heme concentration in MEL cells induced with butyrate with and without SA. MEL cells were grown in RPM1 1640 containing 1% fetal bovine serum. 1 mM butyrate and 0.1 mM SA were present for the entire 7 day heme concentration in cells grown in medium incubation period. o-o: containing 1 mM butyrate. o-o: heme concentration in cells grown in medium containing 1 mM butyrate and 0.1 mM SA.
The heme content with
those
grown
a decreased
Though
inhibition than
fetal
bovine
entered cells
and that
(Table
2),
taken
cells
Fetal
it
Figure
occurred,
uninduced
the the
MEL cells
(Figure
appeared
entire
compared
1.
The SA
7 day period.
levels
observed
grown
in medium
bovine
ALA dehydratase
to inhibit
2).
defined
Furthermore, bovine
because
2 shows the points
kinetics in the
1% fetal unknown
serum were
were
never
containing
1386
l),
that
synthesis
in induced
in Figure
2, the as the protein
was shown to contain bovine
serum)
it
was investigated
gelatin
which
heme might
be
of growth-promoting
by the
growth
curve
it
effects
obviated
of cell growth
shown
medium containing
serum was avoided
(Table
and growth
In the experiments
of medium containing
in fetal
hemoglobin
on heme concentration
in completely
heme/ml
at various
SA inhibited
effect
up by the cells.
substances
cells
its
grown
(79 pmoles
for
was shown that
cells,
source.
observed
seen in Figure
throughout
of heme synthesis
it
were
+ 0.1 mM SA is
of 1 mM butyrate
serum.
in uninduced cells
in the presence
heme concentration
those
After
grown
in 1 mM butyrate
caused
lower
of cells
use of gelatin.
and the
in response
heme concentrations to the continuous
of
Vol.
88,
No.
BIOCHEMICAL
4, 1979
AND
BIOPHYSICAL
RESEARCH
A
4r
COMMUNICATIONS
El
A
j
70
3 -/
2 F
70 c
3
60
i
I
:;4!i 0
FIGURE
3
4
5 DAYS
6
7
8
9
10
2. Growth characteristics and heme concentration in MEL cells grown defined medium with and without SA. MEL cells were grown in modified F-12 medium supplemented with 2.5% gelatin and 30 nM insulin. On days and 7 the cells were subdivided to concentrations of 3~10~ live cells/ml in fresh medium with or without SA as indicated. Growth curves beyond day 3 are corrected to show continuous growth. o-o: cell growth. O-0: heme concentration. Panel A shows the growth pattern and heme concentration of MEL cells in medium without SA. Panel B shows the growth properties and heme concentration of cells grown in the above Panel C shows growth kinetics and heme medium containing 0.1 mM SA. concentration of cells grown in the above medium containing 1 mM SA.
exposure until
of day
increased below
12
50
44
MEL
to
7 at
which
time
cell
counts
until
day
3 and
then
gradually
pmoles
heme/mg
SA.
Control
cells
protein.
In
cells
(panel
began
to
A)
decrease.
decreased. contrast
1387
continued
to
Heme the
untreated
to
divide
Heme
concentrations
levels
never cells,
dropped MEL
in 3
Vol.
88,
No.
cells
4, 1979
exposed
to
experimental
0.1
cells levels
control
in
mM SA (Panel
cell
growth
day
cells.
the
rate
protein,
heme
cell
death
the
increased
cellular l/6
heme
the
lowest
concentrations
the
entire
day
heme/mg
0.1 When
COMMUNICATIONS
for third
pmoles
containing
initially
about
38
sustained.
point
divide
until to
medium
RESEARCH
to
increased
was
which
Cellular of
in
BIOPHYSICAL
continued
decreased
growth
3 at
heme/mg
MEL
cell
B)
levels
slowly grown
C),
on
pmoles
then
cells
levels,
density
Heme
and
AND
mM SA (panel
period.
control heme
8lOCHEMlCAL
as
with
Though
slightly
MEL
cells
were
and
cells
reached
concentrations
below
exposed
decline
1
maximum to
observed
to
to
dropped
concentration
day
the
protein.
mM SA were
continued
10
in
7 untreated
thereafter,
and
increased.
DISCUSSION
The in
MEL
present cells
presented sible
rat
liver
of
the
of
dehydratase
The
effect
in
which
butyrate. by
In
the
lowered With
the
heme
reaches
the
inhibitor of ALA rate
alone
an
increased
synthatase. of
of
heme
at
of
of
to
significantly
induced
SA appeared
to
diminish
the
cells
of
inhibitor
cells
grown
in
3 times
those
seen
of least (Figure
2). size
of
This
increased
biosynthesis
in
reason
for
ALA
caused
by
the
this the
cells
medium
grown is
MEL
such
of
thought
as
even
turn if
as
it
in
the to
and
2). seen
butyrate
and
evidenced
Table is
that
(Figure
1)
presence
of
be
the
result
induction leads
the
cells
DMSO
butyrate-mediated in
great
production.
1 & 2,
the
irrever-
inhibition
synthesis,
presence
cells
1388
agents
be
in
untreated
heme
concentration
induced
heme
be
unpublished
profound
by
in
to
present
(Figures
the
The
ALA
is
both
in
it B.C.,
diminish
was
dehydratase will
demonstrate
in
ALA
inhibition
biosynthesis,
synthesis
concentration
the
tissues
examined
of
of
Frykholm,
most
heme
was
will
R.A.,
in
concentration
pool
kinetics
Hess,
compound
cases,
same
the
enzyme needed
this
heme
inhibition
dehydratase
hemoglobin both
profound
which
D.P.,
rate-limiting is
of
the
publication,
ALA
concentration levels
Details
(Tschudy, Since
excess
those
1).
a subsequent
observations).
ALA
demonstrates
(Table in
in
study
amount
to
a greater of
active
of
Vol.
88,
No.
ALA
dehydratase
model
of
4, 1979
is
this
It
BIOCHEMICAL
is
the
type seen
of
heme
concentration
which
would
approach
tions
(Table
1). does
inhibit
ALA
the
period.
In
heme
the was
shown
MEL
heme
in
experiment
be
present
of In
fetal these
the
bovine
below
heme
biosynthesis,
a)
intracellular
in
which
presumed when in
Figure
concentration
of
inhibition
in
a completely (Figure
10
to
pmoles
should
with
heme the
2 suggest decreases
that
effect cell to
that
by
of
heme
of
the
than
a 3 day was
fact
that at
those
the
possibility
cells,
the
a in that
final
biosynthesis
by
using
by
the
serum
higher
medium
up
over
measurement
MEL
profoundly
taken
one
of
up
In
heme
SA on
gelatin
b)
decrease degradation
in
dilution is
l/6
1389
can
these
on decline from about
cells
heme
by
would cell
lower
to
inhibition
when level
of
of
by
2 mechanisms:
cell
division
50%. be
1
progressive
It
a minor
division.
accelerated the
of
declined
resulting
rapidly
approximately
with
concentration
of
presence
experiments
dilution
in
the
concentrations
these
heme
in
concentration
concentrations
or
death
grown
heme heme
protein.
of
view
Because
cells
in when
degradation,
division
the
but
bovine
in was
defined
shown
intracellular
Either
2).
rapidly
heme/mg
prepara-
measured,
in
that
to be
markedly
cell
constant
serum
taken
decrease
die
not
possible,
1 & 2).
effect
broken
may
fetal
which
be
a pronounced
intracellular
compared
Tables
medium
which
protein)
was
the
bovine
might
it
heme
each
fetal
serum
serum
began
is
than
considered.
approximately
to
cells
heme/mg
experiments
Cells
levels
the
1,
in
SA were
be
medium
in
conducted
mM SA experienced growth.
by
(Figure bovine
was
to
higher
to
concentration
in of
necessary
much
be
A quantitative
(8,9).
dehydratase must
heme
appeared
pmoles
involving
growth
place
heme
cells
fetal
of
involving
of
is
COMMUNICATIONS
cells.
medium
intracellular
traces
medium
the 2)
explanations
or
uninduced
SA in
ALA
concentrations
(269
untreated
cell
to
of
RESEARCH
discussed
(Figure
sufficient
studies
uptake
concentration
the
achieve
the
the
of
cells
possible
and
previously
inhibition
Intracellular in
been
the
BIOPHYSICAL
induced
concentration
100% Two
both has
in
not
concentration
added,
the
dehydratase,
cells.
in
system
that
lower
inhibitor
same
AND
factor The
the
heme
untreated
is
data
Vol. 88, No. 4, 1979
cells.
With
this
level
cell
divisions
a 50% dilution
would
a) diminished decreased of protein cells
for
be reached observed
The killing doublings
BIOCHEMICAL
effect
and normal
division
2 cell
divisions. death
of SA on MEL cells
which
of 1 mM SA could
result
respiration
resulting
synthesis.
synthesis
each cell
when accelerated
in the presence
protein
after
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in several
from
lowered
it
would This
be expected
was the number
of cells
of
occurred. had progressed
through
from 2 possible cytochrome
levels,
Heme has been shown to be involved types
that
(10,11,12),
including
2
mechanisms: and/or
b)
in initiation both
MEL
reticulocytes.
REFERENCES 1.
2. 3. Z:
F: 8. 9. 10. 11. 12.
Lindblad, B., Lindstedt, S., Steen, G. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4641-4645. Gibson, K. Cl., Neuberger, A., Scott, J. J. (1956) Biochem. J. (London) 61, 618-629. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. (1957) J. Biol. Chem. 193, 265-275. Mauzerall, D., Granick, S. (1956) J. Biol. Chem. 219, 435-446. Sassa, S., Granick, S., Chang, C., Kappas, A. (1974) In Erythropoiesis, Proc. of the 4th Int. Conf. on Erythropoiesis. Eds. Nakao, K., Fischer, J. W., Takaku, F., pp. 383-396. Crosby, W. H. and Furth, F. W. (1956) Blood 11, 380-383. Ebert, P. S., Wars, I. M. Buell, D. N. (1976) Cancer Res. 36, 1809-1813. Tschudy, D. P., Bonkowsky, H. L. (1973) Molec. Cell. Biochem. 2, 55-62. Tschudy, D. P. (1973) Molec. Cell Biochem. 2, 63-70. Dabney, 8. J. and Beaudet, A. L. (1978) J. Biol. Chem. 253, 7124-7126. Datta, A., DeHaro, C., Sierra, J. M., Ochoa, S. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3326-3329. London, I. M., Clemens, M. J., Ranu, R. S., Levin, D. H., Cherbas, L. F., and Ernst, V. (1976) Fed. Proc. 35, 2218-2222.
1390