Vol.
3, 865-871,
December
Interaction
inducing
Tokyo
of Applied
Cell Growth
of Two Cytoplasmic
Erythroid
Factors in Mouse
Erythroleukemia
Toshio Watanabe’
Institute
1992
tiated
and Michio Oishi
Microbiology,
University
of Tokyo,
thus
Bunkyo-ku,
113, Japan
Abstrad
Our previous cell fusion experiments have suggested
that the in vitro erythroid differentiation
of mouse
erythroleukemia
cells is the result of a synergistic
readion involving two intracellular differentiationinducing fadors (DIF); these were subsequently
demonstrated in the cytoplasmic fradion of mouse
erythroleukemia
cells. Here, we present experimental
evidence indicating that, under conditions in which the
two fadors (DIF-l and DIF-li) are coinduced, a new
fador, which can trigger erythroid differentiation
upon
introdudion
into undifferentiated
mouse
erythroleukemia
cells, is produced
in the cells. A
similar
fador
was also generated
in vitro after the
incubation
of partially
purified
DIF-l and DIF-Il.
We
found that protein phosphatases could substitute for
DIF-Il. These and other experiments suggest that
protein dephosphorylation
at a tyrosine residue(s) is
involved in the generation of the new fador.
of cell differentiation
has been
in a variety of experimental
sys-
tems,
molecular
of the
cascade,
particularly
that of the early events leading to differentiation,
remains
unknown.
In MEL2 cells, a number
of compounds
trigger
differentiation
into erythroid
cells in vitro (1, 2). One of
the most attractive
hypotheses
advanced
to explain the
manner
in which
these compounds,
with their wide
variety of molecular
structures
and, presumably,
different
biological
functions,
induce
differentiation
is that the
molecular
cascade is quite diverse at the initial stage, but
that it eventually
converges
toward
a common
and critical step for cellular commitment
to differentiation.
We have previously
reported
the presence
of two
proteinaceous
erythroid-inducing
factors (DIF-I and DIFII) in the cytoplasmic
fraction
of MEL cells (3, 4). The
synthesis
of DIF-l
was
induced
following
treatment
with
a DNA replication
inhibitor,
and that of DIF-Il was induced
by most erythroid-inducing
agents, e.g., DMSO
and HMBA. These intracellular
factors triggered
erythroid
differentiation
when
introduced
into undifferentiated
MEL cells, provided
that the recipient
cells were poten-
Received
6/9/92.
1 To whom
requests
The abbreviations
2
entiation-inducing
ylene
dium;
for reprints should be addressed.
used are: MEL, mouse erythroleukemia;
factor;
bisacetamide;
MMC,
FCS, fetal calf serum;
DM50,
dimethyl
sulfoxide;
mitomycin
C; MEM,
PBS, phosphate-buffered
HMBA,
DIF, differhexameth-
minimal
essential
saline.
DifferentiationCells
induction
the
of either
complementary
one
of these
nature
of
factors,
the
two
tons;
DIF-Il,
‘-300,000
daltons).
In this paper, we report experimental
results suggesting
that these two differentiation-inducing
factors (DIF-I and
DIF-Il) interact
with each other and produce
an apparently new erythroid-inducing
factor. The new factor was
distinguished
from the previously
reported
DIF-l and
DIF-Il by its different
chromatographic
behavior
and by
its capability
of triggering
erythroid
differentiation,
by
upon
introduction
into
undifferentiated
‘MEL
cells.
Results
Although
the mechanism
extensively
investigated
nature
865
factors for erythroid
differentiation.
Attempts
to purify
these factors to homogeneity
for cloning
purposes
have
so far been unsuccessful,
mainly owing to the extremely
small quantity
in which they are found in the cells and
the rather time-consuming
and cumbersome
procedures
required
to assay their activity.
Besides their proteinaceous nature, all we know about the biochemical
nature
of these factors
is their behavior
in several
types of
column
chromatography,
the retention
of DIF-I in antiphosphotyrosine
anti body-conjugated
colu mns,
and
their approximate
molecular
sizes (DIF-I, ‘-90,000
dal-
itself,
Introdudion
the
by the
indicating
& Differentiation
me-
Our previous
cell and cytoplast
fusion experiments
(5-7)
suggested
that the erythroid
differentiation
of MEL cells
was a result of the synergistic
action of DIF-l and DIF-Il
and that this probably
involved
interaction
of the two
factors.
To confirm
whether
this was the case, we examined
the chromatographic
behavior,
as well as the
erythroid-inducing
activity,
ofthese
two factors after they
were coinduced
in the cells. For this purpose,
MEL cells
were incubated
in the presence
of a DNA replication
inhibitor,
MMC,
for 18 h (DIF-l
induction).
The
cells
were
then briefly exposed
to DMSO for 6 h (DIF-Il
induction).
The results of our previous
experiments
indicated
that,
under these conditions,
the two factors would reach their
maximal
levels (24 h total incubation
with MMC for DIFI and 6 h incubation
with DMSO for DIF-Il). Continued
incubation
of such treated cells after removing
MMC and
DMSO
for another
4-5 days led most of the cells to
differentiation,
but
MMC
treatment
(24 h) or exposure
to
DMSO (6 h) alone had no effect (data not shown).
Extracts
prepared
from such coinduced
cells were
fractionated
through
a DEAE-cellulose
column
by stepwise salt (NaCI) elutions,
and the erythroid-inducing
activity
in each
fraction
was
assayed.
As shown
in Fig.
1, in
control
experiments,
incubation
with MMC alone for 24
h produced
a factor (DIF-l) that was eluted at 250 mti
NaCI (Fig. 1B), and incubation
with DMSO
for 6 h induced a factor (DIF-Il)
that was eluted
at 50 mi NaCI
(Fig. 1C). Because
of the synergistic
action
of these
factors in erythroid
differentiation,
these two erythroidinducing
factors were detected
only by a complementation assay, in which the recipient
cells were either pulsed
with
DMSO
(DIF-Il
induction)
for
DIF-I
or treated
with
866
Interaction
of Mouse
50
mU
150
4
Erythroid-inducing
mM
250
mM
Factors
A
50
mM
150
mM
250
interacted
B
mM
‘1
was
20
and generated
capable
introduction
into
erythroid-inducing
mM NaCI eluate in
incubated
with high
ii
a new
of inducing
50
mM
130
mU250
50
mM
mM
150
mM
250
D
mM
4
2
I
Since the
vide
further
the possible
10
-
by itself,
upon
h). Under
MEL cells
these conditions,
were committed
a
to
differentiate.
On the other hand, no activity
was found
in the 1 50 mM NaCI eluate of extracts
prepared
from
cells incubated
with DMSO for a prolonged
time in the
presence
of 12-O-tetradecanoylphorbol-1
3-acetate,
a
specific
inhibitor
of MEL cell differentiation,
or in the
eluate of extracts prepared
from differentiation-resistant
MEL cells incubated
with DM50.3
These results support
the view that the activity
in the 1 50 mti NaCI eluate is
closely associated
with MEL cell differentiation.
20
2O30
which,
differentiation
undifferentiated
MEL cells. Similar
activity
was also observed
in the 150
cell-free
extracts prepared
from cells
concentrations
of DMSO (“.‘200 mM)
for a prolonged
time (‘-‘24
substantial
number
of the
C
factor,
erythroid
experiments
information
interaction
described
above
did not proon the biochemical
nature
of
between
the two
factors,
we
attempted
to reconstitute
the process in partially
purified
DIF-l and DIF-Il in vitro. For this purpose,
we incubated
a mixture
of partially
purified
DIF-I and DIF-Il, fractionated the incubated
mixture
on a DEAE column,
and
assayed
Fraction
Fig. 1.
Erythroid.inducing
activity
No.
in cell-free
extracts
of coinduced
cells.
MEL cells (1 1A2; 10 liters culture in each case) were incubated
under the
following
conditions:
A, control;
B, 24-h incubation
with MMC (1 gg/mI);
C, 6-h incubation
with DMSO (1.8%, v/v); D, 18-h incubation
with MMC
(1 ,g/mll
plus
6-h
incubation
with
MMC
(1 sg/mI)
and
DMSO
(1.8%,
v/
v); F, same as D, but assayed under different
conditions
(see below). The
cytosol fraction
(1 20 mg protein)
of each sample was applied
to a DEAE
column (12 x 55 mm) and eluted in a stepwise
manner with 80 ml each
of 50 ms, 150 mM, and 250 msi NaCI in the basal buffer. Twenty gI of
each fraction
(2 ml) were assayed
for erythroid-inducing
activity.
0,
activity assayed with recipient
cells pulsed with DMSO and made permeable (DIF-l activity); #{149},
activity
assayed
with recipient
cells irradiated
with
UV light and made permeable
(DIF-Il activity);
A, activity
assayed with
recipient
cells without
any pretreatment
and made permeable;
& activity
assayed with recipient
cells that were not subjected
to pretreatment
or
to permeabilization.
Erythroid-inducing
activity
is shown as the percentage of benzidine-positive
(81 cells in the total number
of cells examined
(left
side).
UV light (DIF-l
induction)
for DIF-Il.
When
we examined
the extracts
prepared
from
MEL cells in which
the two
factors
had been coinduced
with
MMC
and DMSO,
as
described
above, quite different
patterns
of chromatographic
behavior
and
erythroid-inducing
activity
emerged.
As shown
in Fig. 1D, whereas
DIF-Il activity
(50 mM NaCI eluate) was still present,
DIF-l activity
(250
mM eluate) disappeared
in the coinduced
cells. Instead,
an
apparently
conditions
NaCI
eluate.
new
(DIF-l
factor,
and
Furthermore,
detected
DIF-Il),
under
appeared
we found
that
the
erythroid-inducing
activity
of each
fraction.
After initial failures in experiments
in which the mixture
was incubated
at ‘-30-37#{176}C, we found that incubation
of the mixture
at a lower temperature
(“.‘3-5#{176}C)for a
longer time (‘-3-4
h) resulted
in the appearance
of an
apparently
new erythroid-inducing
factor that was very
both
in the
the factor
assay
150 mi
newly
emerged
in the 1 50 mii NaCI eluate was detected
with
recipient
cells that had not been treated
with UV or
DMSO,
although
it was still necessary
to carry out the
permeabilization
of the recipient
cells to macromolecules
to detect the factor (Fig. 1 E). These results suggest that,
in the coinduced
MEL cells, the two factors somehow
similar
to that found
in the coinduced
Fig. 2C, the factor
was eluted
at 150
cells. As shown
in
mt’i NaCI and was
detected
by DIF-Il or DIF-l assay. As observed
in the
coinduced
cells (Fig. 1), DIF-l activity,
which
was normally eluted at 250 mM NaCI, was greatly reduced
after
incubation,
whereas
almost all of the DIF-Il activity
was
still
recovered
in the
50 mti
NaCI
eluate
(Fig.
2C).
Incu-
bation
of DIF-l or DIF-Il with control
fractions
corresponding
to DIF-Il or DIF-I, respectively,
had no effect
on the elution
pattern of these factors (Fig. 2, A and B).
No
specific
cofactors
were
required
for the
appearance
of the new factor,
as long as the pH of the reaction
mixture was kept between
6.5 and 7.5 (data not shown).
Fig. 3 shows MEL cells (after benzidine
staining)
into
which the 1 50 mM NaCI eluate was introduced
(A) and
the Northern
hybridization
pattern offl-globin
transcripts
from the cells (B).
To elucidate
the biochemical
nature of the reaction
which led to the presence
of this new factor, we exammed a number
of compounds
that might have affected
its generation.
We found that Na3VO4 and ZnCI2, strong
inhibitors
rosine
of protein
phosphatases,
phosphatases
including
protein
ty-
the
reaction.
As
(8, 9), inhibited
shown
in Fig. 4, the presence
of Na3VO4
or ZnCl2
at
relatively
low concentrations
(0.05 mti for Na3VO4 and
0.05 mM for ZnCI2) almost
completely
inhibited
genera-
tion of the new factor.
Since DIF-l and DIF-Il still remained
active after incubation
with these compounds
(Fig. 4, C and D), it appeared
that these compounds
did
not
inactivate
the
with
these
two factors.
3
T. Watanabe,
N. Morita,
erythroid-inducing
activity
The results
and M. Oishi,
suggested
manuscript
associated
that protein
in preparation.
50mM
150mM
B
250mM
Cell Growth
1
4
& Differentiation
4
20
-...0
10
0’’
U)
5
Fraction
10
15
No.
Generation
of a new erythroid-inducing
factor in vitro. The preparation
of partially
purified
DIF-I and DIF-Il is described
under “Materials
and
Methods.”
For control
fractions
corresponding
to DIF-I and DIF-Il, cell-free
extracts,
prepared
from cells which had been treated with neither MMC nor
DMSO, were fractionated
in the same manner as for DIF-l and DIF-Il, and fractions
corresponding
to DIF-l and DIF-Il were pooled.
The mixtures
(total,
1 7 ml) were incubated
at 4”C for 4 h. A, DIF-l (10.8 mg); B, DIF-Il (0.43 mg); c, DIF-l (10,8 mg) and DIF-Il (0.43 mg). For A and B, the incubation
mixture
contained the fractions corresponding
to DIF-Il (A) or DIF-l (8) prepared from control cells, with the amount
of proteins
equivalent
to DIF-Il (0.43 mg) or
DIF-l (10.8 mg). After incubation,
the basal buffer (190 ml) was added to each sample, and the samples were fractionated
through
a DEAE column
(9 x
16 mm) by stepwise
elution with basal buffer containing
50 mba, 150 mxi, and 250 mi NaCI (each 6 ml), respectively.
A portion
(20 MI) of each fraction
was then assayed for erythroid-inducing
activity,
using different
recipient
cells. 0, using recipient
cells pulsed with DMSO and made permeable
(DIF-l
assay); #{149},
using recipient
cells irradiated
with UV light and made permeable
(DIF-Il assay). Erythroid-inducing
activity
is shown as the percentage
of
benzidine-positive
(B’) cells in the total number
of cells examined
(left scale).
Fig. 2.
dephosphorylation
(by a phosphatase)
was somehow
involved
in the generation
of the factor in the 1 50 mti
NaCI eluate.
Several
other
lines of experimental
evidence
supported this view. First, we found that phosphatases
could
substitute
for DIF-Il
(but not for DIF-l)
in the generation
of the new factor. As shown
in Fig. 5, incubation
of DIF-
I with bovine
heart protein
ducing
factor
intestinal
alkaline
phosphatase
or bovine
phosphatase
generated
the erythroid-ineluted
in 150 mi
NaCI with an almost
concomitant
disappearance
of DIF-l. Bacterial
alkaline
phosphatase
also substituted
for DIF-Il (data not shown).
Incubation
with heat-inactivated
phosphatases
or with
these phosphatases
alone did not lead to the generation
of the factor (data not shown).
It appears that the factor
present
in the DIF-Il
preparation
acted on DIF-l in a
manner similar to that of protein
phosphatases,
converting DIF-l into a form that was eluted
at 1 50 mi NaCI.
There was a concomitant
change
in erythroid-inducing
activity,
from that of DIF-l to one which
alone could
trigger
erythroid
differentiation
(without
any pretreatment of recipient
cells). Furthermore,
the presence
of
phosphotyrosine
during
the
incubation
of DIF-l
and
DIF-
II almost
completely
inhibited
the appearance
of this
activity,
whereas
the presence
of either phosphoserine
or phosphothreonine
had negligible
effects (Fig. 6), suggesting that the dephosphorylation
of phosphotyrosine
moieties
was involved
in this process.
Discussion
In this
paper,
we
have
presented
experimental
evidence
to suggest that the erythroid
differentiation-inducing
factors, DIF-I and DIF-Il reported
previously,
interacted
and
produced
a new factor which,
by itself, was capable
of
inducing
the erythroid
differentiation
of MEL cells when
introduced
into the cells. The new factor was produced
in the
cells
when
the
two
other
factors
were
coinduced
and was also produced
in vitro after a mixture
of DIF-l
and DIF-Il was incubated.
Interestingly,
when the new
factor was generated,
DIF-l activity
was lost or greatly
reduced,
suggesting
that the new factor was generated
at the expense
of DIF-l. It is, therefore,
quite likely that
the factor was derived
from a modification
product
of
DIF-I. The in vitro experiments
with partially
purified
DIFI and DIF-ll further
suggest that the reaction
leading to
the generation
of the new factor involved
the protein
dephosphorylation
of DIF-I. This idea is most strongly
supported
by the finding that protein
phosphatases
can
substitute
for DIF-Il. We also present evidence,
although
it is indirect,
that the dephosphorylation
may have occurred at a tyrosine
residue(s)
of DIF-l.
Protein
dephosphorylation
at phosphotyrosine
residues of specific
proteins
has been implicated
in the
commitment
of cell differentiation
primarily
via experiments using inhibitors
of tyrosine
protein
kinases.
We
867
868
Interation
of Mouse
Erythroid-inducing
A
Factors
20
, 1.-i
J”
,,..t
.
;,-,,
‘:‘-Z.
.
e.
‘
.
:
‘
150
mM
250
mM
50
mM
150
L
mM
250
1\
mM
I
10
-.
.
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‘.
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L2
C
B
50
,
mM
150
I
12
mM
250
D
50
mM
I
mM
150
mM
Fraction
fig.
4.
Effects
of Na3VO4
and
ZnCI2
mM
I
5
t-globinE
250
I
.
10
15
No.
on the
generation
of erythroid-
inducing
activity
in vitro.
DIF-l and DIF-Il
after DEAE fractionation
were
concentrated
approximately
2-fold
with
a Minicon
apparatus,
(B-iS;
Amicon).
DIF-t was dialyzed
against the basal buffer
before
concentration.
The samples
were
mixed
in the following
combinations
(total.
3 ml) and
incubated
for 3 h at 4’C with
or without
Na3VO4
(50 gM) or ZnCI2 (50
A, DIF-l
(4 mg protein);
B, DIF-I (4 mg protein)
plus DIF-Il
(0.08 mg
protein);
C, DIF-I (2 ml, 4 mg protein)
plus DIF-II
(1 ml, 0.08 mg protein)
and Na3VO4
(final concentration,
50 zM);
0, DIF-l (4 mg protein)
plus
DIF-Il
(0.08
mg protein)
and ZnCl2
(final
concentration,
SO zM).
After
incubation,
each sample
was dialyzed
against
the basal buffer,
applied
to
a DEAE
column
(3 x 40 mm),
and eluted
stepwise
with
basal
buffer
containing
50 mM, 150 mM, and 250 mxi NaCI (each
3 ml), respectively.
A portion
(20 MI) of each fraction
was assayed
for DIF-l,
DIF-lI,
and the
new factor,
as described
in “Materials
and Methods.”
0, activity
assayed
with
recipient
cells
pulsed
with
DMSO
and made
permeable
(DIF-I
activity);
#{149},
activity
assayed
with
recipient
cells irradiated
with
UV light
and made
permeable
(DIE-Il
activity);
A, activity
assayed
with
recipient
cells without
any pretreatment
but made
permeable.
Erythroid-inducing
activity
is shown
as the percentage
of benzidine-positive
(if’) cells in the
total number
of cells examined
(left scale).
NM).
Fig. .3
trans(ripts
Appearance
of benzidine-positive
by introduction
of the
150
cells and induction
msi
eluate.
MEL
of -globin
)DS19)
cells were
cultured,
and,
when
the cell density
reached
2 x 106/ml,
they
were
permeabilized
to macromolecules
and divided
into two.
One
half was
exposed
to basal I)uffer
(control),
and the other
half was exposed
to the
concentrated
150 m
NaCI eluate (Fig. 2C) (protein
concentration
was
20 mg/mI).
Both samples
were then incubated
at 37’C
in MEM containing
12% FCS. ,-\, the cells were stained
with benzidine
on the fifth day of the
incubation,
and photographs
were
taken
under
a microscope;
B, cytoplasmic
RNAs were isolated
from portions
(2 ml) of each sample
on the
third
day of the incubation
and subjected
to Northern
blotting
using a
#{176}P-labeled !-globin
probe
(pMdGA(
as described
in Materials
and Methods.”
1, permeabilized
and exposed
to basal buffer;
2, permeabilized
and
exposed
ti) the
150 ms
NaCI
eluate.
For details,
see “Materials
and
Methods.”
serine/threonine
kinase
experiments
and others
have reported
that a series of inhibitions
of
protein
tyrosine
kinases
are very effective
inducers
of the
in vitro differentiation
of several
cell lines, including
MEL
cells (10-14).
For example,
herbimycin
A has been found
to induce
the erythroid
differentiation
of MEL cells, as
well
as the embryonal
differentiation
of mouse
embryonal
carcinoma
(F9) cells (11). Erythroid-inducing
activity
has also been observed
with other
tyrosine
kinase
inhibitors,
e.g., synthetic
analogues
of phosphotyrosine,
such as ST638 or methyl-2,5-dihydroxycinnamate,
particularly
when
these
inhibitors
were
combined
with
DNA
replication
inhibitors
such
as MMC
(10, 14). No such
erythroid-inducing
activity
has
been
detected
with
ample,
had
Na3VO4
differentiation
ing
the
typical
inhibitors
results
has
been
of MEL
treatment
erythroid-inducing
with
agents
such
all of the
phosphotyrosine-containing
were
reduced
or had disappeared
of
with
that
differentiation
in specific
with
(15).
the experimental
dephosphorylation
the
entiation
cellular
reaction(s)
of MEL
These
for
the
erythroid
1 5). Furthermore,
cells
proteins
probably
responsible
cells.
or
as HMBA,
are
all
results
presented
here,
of a phosphotyrosine
for
ex-
follow-
DMSO
proteins
at a very early
results
other
these;
to inhibit
(14,
MEL
14). Several
with
found
cells
of
(13,
consistent
other
almost
either
stage
consistent
suggesting
residue(s)
is closely
associated
triggering
the
differ-
Cell Growth
A
20
50
mM
150
mM
250
& Differentiation
869
B
50
mM
mM
150
mM
250
mM
10
V
D
20
50
mM
150
mM
250
mM
50
mM
150
I
mM
I
250
mM
I
10
0
5
10
15
5
Fraction
10
15
10
5
No.
15
5
Fraction
Effects of phosphatases
on the generation
of erythroid-inducing
factor in vitro. DIF-l and DIF-Il were prepared
as described
in “Materials
and Methods.”
DIF-l was dialyzed
against the basal buffer for 16 h and
concentrated
approximately
2-fold with a Minicon
apparatus
(B-15; Amicon). DIF-Il was also concentrated
with a Minicon
to approximately
5fold its original concentration.
Alkaline
phosphatase
and protein
phosphatase were dialyzed
against basal buffer containing
50 mwi NaCI for 16
h before use. The samples
were mixed in the following
combinations
(total, 2.2 ml) and incubated
for 3 h at 4”C. A, DIF-l (5 mg protein);
B,
10
15
No.
Fig. 5.
DIF-l (5 mg
protein)
plus DIF-lI (0.1 mg
protein); C, DIF-l (5 mg
protein)
plus alkaline
phosphatase
(from bovine intestinal
mucosa, 440 units); 0,
DIF-l (5 mg protein) plus protein phosphatase
(from bovine heart muscle,
44 lag). After incubation,
basal buffer (15 ml) was added to each sample.
The samples
were applied
to DEAE column
(3 x 30 mm) and eluted
stepwise with the basal buffer, containing
50 m,i, 150 msi, and 250 mxi
NaCI (each 3 ml), respectively.
A portion
(20 gI) of each fraction
was
assayed for DIF-I, DIF-Il, and the new factor, as described
in “Materials
and Methods.”
0, activity assayed with recipient
cells pulsed with DM50
and made permeable
(DIF-l activity)
#{149},
activity
assayed with recipient
cells irradiated
with UV light and made permeable
(DIF-ll activity);
A,
activity assayed with recipient
cells without
any pretreatment
but made
permeable.
Erythroid-inducing
activity
is shown
as the percentage
of
benzidine-positive
(ff’i cells in the total number
of cells examined
(left
scale).
Materials
and
Methods
Materials.
L-a-Lysophosphatidylchohne
(lysolecithin)
was purchased
from Sigma (St. Louis, MO), and MMC
was obtained
from Kyowa Hakko (Tokyo, Japan). HMBA
was a generous
gift from Dr. 1. Yamane.
All of the other
chemicals
used
were
reagent
grade.
Eagle’s
MEM
was
obtained
from Nissui Seiyaku (Tokyo, Japan). Ham’s nutrient
mixture
F-12 and Dulbecco’s
modified
(Eagle’s
medium)
were
purchased
from Sigma,
and FCS was
obtained
from
Flow
Laboratories
(McLean,
VA) and
United
Biotechnologies
(Tokyo,
Japan). Sodium
orthovanadate
(Na3VO4) was purchased
from Aldrich
Chemi-
of phosphoamino
acids on the generation
of erythroidin vitro. The DIF-l and DIF-Il fractions
were prepared
as
described
in Materials
and Methods.”
DIF.l was dialyzed
against the
basal buffer for 16 h and concentrated
approximately
2-fold in a Minicon
(B-iS; Amicon).
DIF-I (5.6 mg protein)
and DIF-Il (0.1 mg protein)
were
mixed,
and the mixtures
(4 ml) were incubated
for 3 h at 4”C in the
presence
of one of the following
phosphoamino
acids. A, control;
B,
phosphoserine
(5 mM); C, phosphothreonine
(5 mM); 0, phosphotyrosine
(5 mM). After incubation,
basal buffer (15 ml) was added to each sample,
and the samples were applied to a DEAE column
(3 x 30 mm) and eluted
stepwise
with the basal buffer containing
50 mi, 150 mxi, and 250 msi
NaCI (each 3 ml), respectively.
A portion
(20 gI) of each fraction
was
assayed for DIF-I, DIF-Il, and the new factor, as described
in “Materials
and Methods.”
0, activity assayed with recipient
cells pulsed with DMSO
and made permeable
(DIF-I activity);
#{149},
activity
assayed
with
recipient
cells irradiated
with UV light and made permeable
(DIF-Il activity);
A,
activity assayed with recipient
cells without
any pretreatment
but made
permeable.
Erythroid-inducing
activity
is shown as the percentage
of
benzidine-positive
(Bi
cells in the total number
of cells examined
(left
Fig. 6.
inducing
Effects
factor
scale).
cal Co. (Milwaukee,
WI). Bovine intestinal
mucosal alkaline phosphatase
was purchased
from Sigma, and bovine
heart muscle phosphatase
was a generous
gift from Dr.
H. Murofushi.
Cells and Cell Culture.
were
generously
Rifkind,
and
MEL (Friend) cells (745A,
provided
P. A. Marks.
by
The
Drs.
MEL
M.
cell
Terada,
line,
11A2,
DS1 9)
R. A.
used
for the preparation
of erythroid-inducing
factors,
was
established
in this laboratory
(4). All cells, except for MEL
11A2, were cultured
in MEM supplemented
with 12%
FCS; the MEL 11A2 cells were cultured
in F-12-Dulbecco’s modified
Eagle’s medium
(1:1, v/v) supplemented
with 2% FCS. All cultures
were incubated
at 37#{176}C
in a
humidified
atmosphere
containing
5%
CO2
in air.
870
Interaction
of Mouse
Partial
Erythroid-inducing
Purification
Factors
of DIF-l
purification
of DIF-l and
scribed
previously,
with
and DIF-Il.
DIF-Il
slight
The partial
was performed
modifications
as de(4). MEL
1 1A2 cells were cultured
in 10-liter
spinner
flasks (total,
-10-20
liters).
When
the cell density
reached
2.5 X 106
cells/mI,
the culture
was diluted
with
fresh medium
to
106 cells/mI
For the preparation
of DIF-I, MMC
was then
added, to a final concentration
of 1 zg/mI, and the culture
was continued
for 24 h. For DIF-Il, DMSO was added to
a final concentration
of 1 .8% (v/v, 280 mM), and the
culture was incubated
for 6 h.
The following
procedures
were used for the preparation of both DIF-l and DIF-Il, unless otherwise
specified.
The cells were collected
by centrifugation,
washed twice
with
PBS (137 mM NaCl-2.4
1.1 mM KH2PO4)
and once
mi
KCI-9.6
mti Na2HPO4with TKM buffer
(10 mri Tris-
Cl, pH 7.5-10
mM KCI-1.5
mi’st magnesium
acetate-0.1
mM dithiothreitol-0.1
mi phenylmethylsulfonyl
fluoride),
and resuspended
in TKM buffer at 5 x 108 cells/mI.
After
being left to stand for 1 5 mm at 0#{176}C,
the cells were
disrupted
with either a Dounce
or a Teflon homogenizer
(Potter-Elvehjem
type, luchi Level 90). The sample was
then mixed with 0.25 volume
S buffer (100 mti Tris-CI,
pH
7.5-1.25
M
sucrose-25
mi
magnesium
acetate
and
centrifuged
first at 1,200 x g for 5 mm, and then at
120,000 x g for 90 mm. The supernatant
(cytosol) fraction
(-500
mg protein)
was diluted
3-fold with basal buffer
(20
mri
Tris-CI,
pH
7.5-10%
thiothreitol)
and applied
x 100
For DIF-l,
mm).
(v/v)
glycerol-0.25
mrsi di-
to a DEAE-cellulose
the
column
was
column
(20
with
150
washed
ml each of the basal buffer,
the basal buffer
50 mM NaCI, and the basal buffer containing
NaCI.
DIF-l
was
then
eluted
buffer containing
250 mst
was first washed with 1 50
factor was eluted with 1 50
mM NaCI. Fractions which
250
mi
eluate
and
DIF-Il
with
150
ml
containing
150 mi
of
the
basal
NaCI. For DIF-Il, the column
ml of the basal buffer, and the
ml of the buffer containing
50
exhibited
activity (DIF-l in the
in the
50
mi
eluate)
were
pooled and dialyzed
against the basal buffer for 6 h. The
samples were concentrated
with a Minicon
apparatus
(B1 5; Amicon)
when
necessary.
Protein
concentrations
were determined
with a protein
assay kit (Bio-Rad).
All
manipulations
were carried
out at 0-4#{176}C,unless otherwise specified.
Assay for
Erythroid-inducing
Adivity.
Assays
for the
erythroid-inducing
activity
of DIF-l and DIF-Il were performed
in essence by a procedure
described
previously
(4, 16). MEL (DS19) cells were cultured
in MEM (supplemented
with 12% FCS) in plastic Petri dishes (60 x 12
mm) at 37#{176}C.
The cells grown to confluence
were collected
by centrifugation
(500 x g for 5 mm), washed
once with PBS, and resuspended
in PBS at 5 x 106 cells/
ml. For the DIF-I assay, 2 volumes
of fresh MEM medium
(with 1 2% FCS) containing
DMSO (420 mM) were added
to the cell suspension
(cell density,
2 x 106 cells/mI),
and
the
cells
were
incubated
for 6 h. The
cells
(total,
5 X 106)
were then collected
by centrifugation
(500 x g for 10
mm) and washed twice with cold PBS. To the sedimented
cells, 1 ml of cold (0#{176}C)
i-a-lysophosphatidylcholine
solution
(4.2 zg/ml
in MEM)
were
thoroughly
mixed
using
was added,
a Pasteur
and
pipet.
the cells
The cell
suspension
was incubated
for 3.5 mm at 0#{176}C,
and then
10 l were quickly
transferred
with an automatic
pipet
to each well of a microplate
(96 wells; Falcon) containing
180 I of prewarmed
(37#{176}C)
MEM with 12% FCS and 20
zI of samples
adjust
the
for assay.
length
poration
of proteins
the
were
cells
assaying
Sometimes,
of the
it was necessary
incubation
for the
(for details,
incubated
to
optimal
incor-
see Ref. 1 6). After
mixing,
at 37#{176}C
in a CO2
incubator.
For
DIF-ll,
cells grown to confluence
were collected
by centrifugation
(500 x g for 5 mm), washed
once with
PBS, and resuspended
in PBS at 5 X 106 cells/mI.
Two ml
of the sample were then transferred
to a plastic Petri dish
(60-mm
diameter)
and irradiated
(20 J/m2) under a germicidal
UV lamp (GL15, 15 W; Toshiba).
After centrifugation, the cells were resuspended,
at 8 x 1 55 cells/mI,
in MEM containing
12% FCS and incubated
for 15 h at
37#{176}Cin a CO2 incubator.
The cells (total,
5 X 106) were
then processed
for permeabilization
as described
above
for DIF-I.
Erythroid
differentiation
was
assayed
on the
fifth
day
of incubation,
by counting
hemoglobin-accumulated
cells (benzidine-positive
cells) stained with benzidine,
as
described
by Orkin et a!. (17).
Northern
Hybridization.
MEL cells (2 ml culture)
were
harvested,
and
cytoplasmic
RNA
was
prepared
as de-
scribed by Favaloro et a!. (18). The RNA (20 zg) was then
electrophoresed
and blotted according
to the procedure
ofGoldberg
(1 9). Cloned mouse fi-globin
DNA (pMGA),
used here as probes, was generously
supplied
by Dr. T.
Yamashita
and nick-translated
by the procedure
of Rigby
et a!. (20) using [32P]dCTP
(specific
activity,
3000 Ci/
mmol; ICN).
Acknowledgments
We wish to thank
S. Nomura
and
T. Kobayashi
S. Yamagoe
for editing
for
their
the manuscript.
valuable
We thank
discussions
Drs.
throughout
the
study.
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