Vol. 189, rjo. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1639-l 644
December 30, 1992
REGULATION OF THE EXPRESSION OF LAMINS A AND C IS POST-TRANSCRIPTIONAL IN P19 EMBRYONAL CARCINOMA CELLS
Jacqueline
LANOIX,
Daniel
Institut
Received
November
20,
SKUP, Jean-Francois
du cancer Montreal,
COLLARD, and Yves RAYMOND
de Montreal, Hopital Notre-Dame, Quebec H2L 4M1, Canada
1992
The polypeptide composition of the nuclear lamina can display important variations: undifferentiated cells express only lamin B and they acquire lamins A and C only after differentiation. We have analyzed the expression of lamins A and C in P19 pluripotent mouse embryonal carcinoma cells. Undifferentiated P19 cells are completely devoid of lamins A and C. We show that undifferentiated P19 cells contain low, but detectable steady-state levels of RNAs for lamins A and C that begin to increase by 24 h of retinoic acid-induced differentiation. However, the rate of transcription of the lamin A and C gene(s), analyzed by runon transcription assays, remains unchanged during the differentiation process. These results demonstrate that, at least in P19 embryonal carcinoma cells, regulation of the expression of lamins A and C is a post-transcriptional event. 0 1992AcademrcPress, 1°C.
Lamins
A,
B and C are
lamina,
the proteinaceous
present
in most mammalian
are present other
quantitative, (394)
cell
types
in their
(3-6),
structural
network (for
components
apposed
review,
amounts
display
lamin
constitutively
lamins
cells
equimolar
mouse EC cells
9
(2,7,8)
major
fibrillar
in roughly
mammalian
the
to the inner
see (1)).
for
variations,
Thus,
and a number
of murine
express
lamin
B, but
early
lack
both
membrane
polypeptides (2);
however,
qualitative
developing
and
mouse embryos
and human tumor
or express
nuclear
nuclear
in HeLa cells
complement.
the
These three
example
important
of
very
cell
lines
low levels
of
accompanied
by
A and C. Appearance
of a more differentiated
the appearance
of lamins
synthesis
lamins
of
promyelocytic or granulocytic
A and C. A
leukemia pathways
and cells
C are
(9,lO).
phenotype
While
expression
shows
a
induced Similarly,
Abbreviations: DMSO, Me,SO, dimethylsulfoxide; glyceraldehyde-3-phosphate dehydrogenase; sulfate.
1639
dramatic
is generally of lamin increase
to differentiate an increased
EC, embryonal RA, retinoic acid;
B remains when along
the
expression
unchanged, HL-60
human
macrophage of
lamins
carcinoma; GAPDH, SDS sodium dodecyl
0006-291 X/92 $4.00 Cop>lright 0 1992 by Academic Prex. Irrc. Ail rights of reproduction in atty form rerervd.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUillCATIONS
Vol. 189, No. 3, 1992 A and C accompanies The EC cell
differentiation
lines
expression
of lamins
A and C also
display
expressed
only
Moreover,
increased
changes of
lamins
A
It
has
C is
with
been
demonstrated
level
This
(9).
of
these
begun
not
(8).
only
on
and
Lamins
development,
in
mouse
v-ras"-induced
Thus,
it
cell
type,
being
embryos
(3).
phenotypic
seems that but
expression also
on
the
cells. that
correlated in the
quantitative
in HL60 cells synthesis
dependent
(3-5).
embryonic
has
(11).
cells state
differentiation
during
cells
U937 tumor
undifferentiated
A and C accompany
cancer
of the
changes
following
differentiation of lamins
F9 cells
Moreover,
correlate
expression
tissue
state
undifferentiated (6).
differential
human lung
and
B in their
occurs
levels
in NCI-H249
lamin
A and C only
after
differentiated
of human monoblastoid
P19 and F9 express
with
rate
lack
of
the absence
of synthesis
variations
prompted is
lamins
the
regulated
A
and
C
of the corresponding
of lamins
in the abundance
correlation
polypeptides
the
mRNAs
A and C were shown to
of their
authors
primarily
in
respective
to conclude at
the
mRNAs that
the
transcriptional
(9).
In this cells
study,
(12,13).
muscle
These
in response
RA as aggregates treated
with
do
express
not
we have analyzed cells
have
to DMSO (13)), (13),
or
lamins
into
into
RA as monolayers
the
In their
C;
however,
We show that
regulation
(4).
C occurs
at a post-transcriptional
to
neurons
(14).
A and
expression
potential
fibroblast-
differentiation mainly
the
of
lamins
differentiate
and glial
cells
and smooth they
state,
these
these
expression
lamins of lamins
P19
cardiac
when treated cells
acquire
of the
into
muscle-type
undifferentiated
level
A and C in
with when cells during A and
in P19 cells.
MATERIALS AND METHODS Cell
culture and materials P19 cells were cultured and differentiated by treatment with RA on monolayers or on with DMSO as described aggregates or in (15). Immunofluorescence and immunoblotting detection of lamins A and C in P19 cells were performed as previously described for F9 cells (5,16). RA was purchased in 95% ethanol (IBI) and from Kodak; a stock solution of 10e3 M was prepared a-[32P]-dCTP (800 Ci/mmol) was purchased kept at -20°C for no more than a week. from ICN and a-[32P]-GTP (3000 Ci/mmol) from Amersham Corp. (Arlington Heights, vanadyl-ribonucleoside complex was purchased from Bethesda Research IL); Laboratories. RNA blot analysis Isolation of cytoplasmic RNA was as in (17). Poly(A)' RNA was fractionated on 1.2% agarose-2.2 M formaldehyde gel (18), with 10 m M phosphate (pH 6.5) as running buffer. Hybridization was carried out at 42°C overnight. The probe was a plasmid bearing the entire coding sequence of murine lamin C (19) kindly provided by Dr. D. Werner (German Cancer Research Center, Heidelberg, Germany). After stringent washes, the blot was exposed to Kodak XRP film. Nuclear
run-on transcriotion assavs Nuclei were isolated as described in (17). assays were carried out essentially as described
1640
Nuclear in (20),
run-on transcription with modifications.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 189, No. 3, 1992
Nuclei (100 pi/assay), isolated as described above, were allowed to thaw on ice and mixed with 100 ~1 of reaction buffer (10 m M Tris pH 8.0, 5 m M t$Clz, 300 m M KCl, 0.5 m M each of ATP, CTP and UTP, 10 /&l GTP, and 200 &i of a-[ PI-GTP (300 Ci/mmol, Amersham)). Reaction was incubated at 27°C for 30 min, at the end of which each reaction received 200 ~1 of 10 m M Tris pH 8.0, 1 m M EDTA, 200 m M vanadyl-ribonucleoside complexes, 25 pg/ml tRNA, 10 mg/ml RNase-free DNase (BRL), was passed a few times through a syringe (18 l/2 gauge), and incubated 30 min at room temperature, followed by proteinase K (75 pg) treatment in the presence of The labeled RNA was purified by 2 phenol-chloroform1% SDS, at 37°C for 1 h. isoamyl alcohol extractions, chloroform extraction and ethanol precipitation. RNA was resuspended in 100 ~1 of sterile Hz0 and passed through a Sephadex G-50 column. Radioactivity in each reaction was determined by Cerenkov counting. In each filter was hybridized with the same number of cpm a given experiment? (usually 0.5 - 1x10 cpm) of labeled RNA. To prepare filters for hybridization, 5,ug of plasmid (pGEM), or plasmid containing cDNA for murine lamin C, described above, or for actin or GAPDH genes were denatured by incubation with 0.2 M sodium hydroxide for 20 min at room temperature, and neutralized with 3 volumes of 50 m M Tris pH 8.0, 1 M NaCl, 50 m M EDTA and 15 volumes of 333 m M Tris pH 6.8. DNAs were spotted onto Biotrans nylon membrane (ICN) using a Biorad slot blot apparatus, UV cross-linked for 2 min and baked at 80°C for 90 min. Filters were pre-hybridized for 6 h at 42°C in 50% deionized formamide, 5xSSPE (1xSSPE = 0.15 M NaCl, 10 m M Na PO,H,O, 1 m M EDTA), 5 x Denhardt's solution (21), 100 ,ug/ml yeast tRNA, 1 m M NaPP6,, 0.1% SDS. At the end of 6 h, buffer was replaced with freshly-prepared buffer and labeled RNA was added. Hybridization was carried out at 42°C for 40-48 h. Filters were washed with 2xSSPE, 0.1% SDS 2x5 min at room temperature, 3x30 min at 55"C, followed by a wash with 0.1 SSPE, 0.1% SDS for 30 min at 65°C. Filters were then treated with lOpg/ml RNase A and 15,000 u/ml of RNase Tl in 2xSSC, at 37°C for 30 min, followed by a final rinse at 37°C in 2xSSC for 15 min. Filters were exposed to Kodak XAR films for a maximum of 2 weeks. RESULTS AND DISCUSSION The kinetics during
differentiation
P19 cells treatment
(Fig.
treatment
RA for
These
8).
materials
RNAs were
the entire
coding
hybridize
with
extensive
portion
Fig. to
mRNAs for of their
at
24 h of
differentiated
along
murine
both
lamins
(lane
6),
RA treatment
abundant
those phorbol
for esters
cultures
of
3),
24 (lane
P19 cells
RA on aggregates
lamin
C (19).
present
at
Levels
1).
(lane than
low
of both
4)
probe
(lanes
6-8).
those
lamin
A.
lamin
A
(9). 1641
when In
HL60
addition,
lamins
share
an
Indeed,
kb corresponding
detectable
levels
gradually
a maximum
pathways for
therefore
(22,23).
and 2.1
species
under
containing
should
two
but
and reach
7) or DMSO (lane
plasmid
sequences 3.0
following
as described
these
and coding
of approximately
following
cells (lane
This
A and C since
from
4) and 72 h (lane
differentiated
and hybridization,
each of the three
more
than
fully
are
C mRNAs were more abundant with
from
5' non-coding
lamin
differentiate
8 (lane
determined
RNA was isolated
used was a nick-translated
for
A and C, respectively, PI9 cells
2),
by blotting
two mRNA species
undifferentiated starting
(lane
The probe
sequence
1 shows that lamins
extracted
analyzed
and methods.
poly(A)'
monolayer
1, lane
RA on monolayers
A and C mRNAs were first
Cytoplasmic
1) and from
2 (Fig.
RNA was also
with
of lamins
of P19 cells. 1, lane
with
Poly(A)'
5).
of accumulation
in
At all
in
increase cells time
full,y points,
Lamin C RNAs were also cells the
were time
induced
course
of
to RNA
Vol.
189,
No.
BIOCHEMICAL
3, 1992
AND BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RA
,,,,“2~EH Lamin
A w
Lamin
C *
kb
RA
\
I
_ 7.5 - 4.4 _ 2.4
12
0
48
72 h
_ 1.4
pGEM w Actin w GAPDH w
01
0
2
GAPDH w
Lamin A/C
w
Fiqure 1. Time course of accumulation of lamins A and C mRNAs in differentiatinq P19 cells. Poly(A)' RNA was isolated from undifferentiated P19 cells (lane 1) and from monolayer cultures of P19 cells treated with RA for 2 (lane 2), 8 (lane 3), 24 (lane 4) and 72 h (lane 5), or 7 days (lane 6). Poly(A)' RNA was also extracted from fully differentiated cells that were treated as aggregates with RA (lane 7), or with DMSO for 10 days (lane 8). Each lane received 5 pg of poly(A)' RNA. RNAs were fractionated on an agarose-formaldehyde transferred to a nylon membrane and hybridized with a nick-translated gel, plasmid containing the entire coding sequence for murine lamin C (19). The membrane was stripped of probe and rehybridized with a nick-translated plasmid containing a GAPDH cDNA insert. Run-on transcriotion assav with nuclei isolated at different staqes Fiqure 2. of RA-induced differentiation of P19 cells. Nuclei were isolated from cells used in the experiment described in Fig. 1: undifferentiated P19 cells, monolayer cultures of P19 cells treated with RA for 12, 48, 72 h, as indicated above each lane. Run-on transcription assays were as described under materials and methods, labeled RNAs were hybridized for 48 h with plasmid containing cDNA inserts (5 kg) immobilized on nylon membranes. Positive (GAPDH and actin) and negative (pGEM) controls are shown.
accumulation
in
A and C accumulation detected
total
in F9 cells,
24 h after Expression
loaded
P19 cells
differentiating
the
onset
of the
in each lane accumulation
in which
increased
of differentiation
the time
to
amounts
Taking
1).
lamins
into
course
of both
of lamin
lamins
were
(5).
GAPDH gene was used as a control
(Fig. of
is similar
account
for
the
the
levels
A and C mRNAs seems more
amounts
of RNA
of GAPDH RNA, the
important
at later
times
of RA treatment. The
presence
undifferentiated number
of
A and C.
of P19 cells
spontaneously Both
not
differentiated
of
mRNAs
be attributed cells
to
P19 cell
confirmed
lamins the
expressing
and immunoblotting (22)
for
the
cultures
(data
sharp
increase
complete not
presence
a high
with
A level
and of
C
in
a small
of
lamins
a highly
specific
lamin
absence
of lamins
A and
shown),
as previously
shown in
(3,4).
To determine was a consequence nuclei
could
antibody
C in undifferentiated studies
amount
small
immunofluorescence
A and C monoclonal other
a
were isolated
whether of
the
transcriptional from
the cells
activation used in the 1642
observed of
in
the
RNA blotting
lamins
lamin
A and C mRNAs
A and C gene(s), experiment
(Fig.
1)
Vol.
189,
No.
and subjected counts
BIOCHEMICAL
3, 1992
to
from
run-on
each
recombinant
transcription
reaction)
plasmids of lamin
cDNA insert
used contains exists
splicing
of
the
transcription
the
that
lamins
of these
pattern
starting
C mRNAs may
unchanged the
the
during for
of lamin C.
in
the
first
lamins
C since
the
However,
the
by
Fig.
2,
alternate
the
three
pattern
of the
experiment,
be produced
transcription
onset
this
lamin
As shown
of mRNAs specific
at 24 h after
that of
RNAs (equal
insert-containing In
sequence
(22,23,24).
Therefore,
of accumulation
increase
A and
denatured from
COMMUNlCATiONS
labeled
filters.
coding
RNAs remains
process.
to
nylon
be distinguished entire
RESEARCH
Extracted
hybridized on
same transcript
differentiation the
were
A cannot
BIOPHYSICAL
assays.
immobilized
transcription possibility
AND
rate
days of the
does not
A and C which
differentiation
of
reflect showed an
process
(Fig.
1). Taken C is
not
cells.
together,
regulated Instead,
responsible
these
for
is
in
gene expression
known that the
RA-induced normally
which
degrade
processing,
to
lamins or
progresses.
lamins is
result
RNA and protein
studies
altered
of
synthesized
a newly
either
A and
C RNAs.
transport
mechanism
is
of
changes
of
proteins.
stability
24 h after of these
a polypeptide
RNAs,
that
polypeptide
lamins
time.
by dramatic
would
that
Alternatively,
experiments
was not addressed
on the
confers
a more
rapid
A and C RNAs may occur
are
in particular
apparent cell
of lamins
A and C was mainly
the
of regulation
concerning
under
as
way to discriminate
directly
level
in these
studies
lines of the
since
Therefore,
our results
are the first
in
expression
of
A and C is
lamins
correlation
between
levels
that
regulation
to conclude
transcriptional
was performed. transcriptional
P19
any given
expression
of an increased of
A and of
A and C mRNAs starting
synthesis
had relied
synthesized
of the expression
P19 cells,
A and C at
accompanied
in
of lamins
Further
of lamins
differentiation
possibilities.
Previous
question
or that
polyadenylation these
process
the
expression
during
a post-transcriptional of
is the result in
them,
stability
that
ultimately
accumulation
that
level
synthesis
the differentiation
modulation
differentiation
demonstrate
suggest the
differentiation
a greater
between
results
increased
reflecting
results
transcriptional
regulating
It
Perhaps
these at the
(9).
synthesis
no run-on
of
lamins
transcriptional
demonstration mostly
However,
regulated
of the
A and C assay
that, at
at least a post-
level. ACKNOWLEDGMENTS
We thank Dr. F. McKeon for a generous gift of anti-lamin AC monoclonal antibody, Dr. D. Werner for a gift of the murine lamin C plasmid, P. Hince for technical assistance, R. Duclos for photography and C. Nault for typing the manuscript. J.L. was supported by a fellowship from the Cancer Research Society, J.F.C. by studentships from the Quebec Ministry of Education and Universite de Montreal, and Y.R. by a chercheur-boursier award from the Fonds de la recherche en Sante du Quebec. This work was supported by grants from the Medical Research Council of Canada.
1643
Vol.
189,
No.
BIOCHEMICAL
3, 1992
AND BIOPHYSICAL
RESEARCH
COMMUNICATIONS
REFERENCES
1. 2. 3. 4. 5.
L. and Burke, B. (1988) Annu. Rev. Cell Biol. 4, M.N., Bensussan, A., Bourge, F.F., Bornens, M. and (1987) EMBO J. 6, 3795-3799. Rober, R.A., Weber, K. and Osborn, M. (1989) Development Stewart, C. and Burke, B. (1987) Cell 51, 383-392. Lebel, S., Lampron, C., Royal, A. and Raymond, Y. (1987)
Gerace, Guilly,
335-374. Courvalin, 105,
J.C.
365-378.
J. Cell.
Biol.
105, 1099-1104. 6.
Worman,
H.J.,
Lazarides,
I. and Georgatos,
S.D.
(1988) J. Biol.
them.
263,
12135-12141. 7.
Paulin-Levasseur, M., Scherbarth, A., Traub, U. and Traub, P. (1988) Eur. Biol. 47, 121-131. R. and Shaper, J.H. (1991) Cancer Res. Kaufmann, S.H., Mabry, M., Jasti, 51, 581-586. Paulin-Levasseur, M., Giese, G., Scherbarth, A. and Traub, P. (1989) Eur. J. Cell Biol. 50, 453-461. Collard, J.-F., Senecal, J.L. and Raymond, Y. (1992) J. Cell Sci. 101, 657-670. Hass, R., Giese, G., Meyer, G., Hartman, A., D&k, T., Kohler, L., Resh, Eur. J. Cell Biol. 51, 265K., Traub, P. and Gopelt-Strube, M. (1990) 271. McBurney, M.W. and Rogers, B.J. (1982) Dev. Biol. 89, 503-508. E.M.V., McBurney, M.W., Jones-Villeneuve, Edwards, M.K.S. and Anderson, P.J. (1982) Nature 299, 165-167. Jones-Villeneuve, E.M.V., McBurney, M.W., Rogers, K.A. and Kalnins, V.I. (1982) J. Cell Biol. 94, 253-262. Rudnicki, A. and McBurney, M.W. (1987) In Teratamas and embryonic stem IRL Press, cells. A practical approach. E.J. Robertson (ed), pp. 19-49, Oxford, UK. Collard, J.-F. and Raymond, Y. (1990) Exp. Cell Res. 186, 182-187. Lanoix, J., Belhumeur, P., Lussier, M., Royal, A., Bravo, R., and Skup, D. (1991) Cell growth and differentiation 2, 391-399. Maniatis, T., Fritsch, E.F. and Sambrook, J. (eds). (1982) Molecular cloning. Cold Spring Harbor Laboratory, New York. Biophys. Acta 1008, 119-122. Riedel, W. and Werner, D. (1989) Biochim. Greenberg, M.E. and Ziff, E.B. (1984) Nature 311, 433-438. Denhardt, D.T. (1966) Biochem. Biophys. Res. Commun. 23, 641-646. McKeon, F.D., Kirschner, M.W. and Caput, D. (1986) Nature 319, 463-468. Fisher, D.Z., Chaudhary, N. and Blobel, G. (1986) Proc. Natl. Acad. Sci. USA 83, 6450-6454. Stick, R. (1992) Chromosoma 101, 566-574.
J. Cell
8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18. ii: E: 23. 24.
1644
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1639-l 644
December 30, 1992
REGULATION OF THE EXPRESSION OF LAMINS A AND C IS POST-TRANSCRIPTIONAL IN P19 EMBRYONAL CARCINOMA CELLS
Jacqueline
LANOIX,
Daniel
Institut
Received
November
20,
SKUP, Jean-Francois
du cancer Montreal,
COLLARD, and Yves RAYMOND
de Montreal, Hopital Notre-Dame, Quebec H2L 4M1, Canada
1992
The polypeptide composition of the nuclear lamina can display important variations: undifferentiated cells express only lamin B and they acquire lamins A and C only after differentiation. We have analyzed the expression of lamins A and C in P19 pluripotent mouse embryonal carcinoma cells. Undifferentiated P19 cells are completely devoid of lamins A and C. We show that undifferentiated P19 cells contain low, but detectable steady-state levels of RNAs for lamins A and C that begin to increase by 24 h of retinoic acid-induced differentiation. However, the rate of transcription of the lamin A and C gene(s), analyzed by runon transcription assays, remains unchanged during the differentiation process. These results demonstrate that, at least in P19 embryonal carcinoma cells, regulation of the expression of lamins A and C is a post-transcriptional event. 0 1992AcademrcPress, 1°C.
Lamins
A,
B and C are
lamina,
the proteinaceous
present
in most mammalian
are present other
quantitative, (394)
cell
types
in their
(3-6),
structural
network (for
components
apposed
review,
amounts
display
lamin
constitutively
lamins
cells
equimolar
mouse EC cells
9
(2,7,8)
major
fibrillar
in roughly
mammalian
the
to the inner
see (1)).
for
variations,
Thus,
and a number
of murine
express
lamin
B, but
early
lack
both
membrane
polypeptides (2);
however,
qualitative
developing
and
mouse embryos
and human tumor
or express
nuclear
nuclear
in HeLa cells
complement.
the
These three
example
important
of
very
cell
lines
low levels
of
accompanied
by
A and C. Appearance
of a more differentiated
the appearance
of lamins
synthesis
lamins
of
promyelocytic or granulocytic
A and C. A
leukemia pathways
and cells
C are
(9,lO).
phenotype
While
expression
shows
a
induced Similarly,
Abbreviations: DMSO, Me,SO, dimethylsulfoxide; glyceraldehyde-3-phosphate dehydrogenase; sulfate.
1639
dramatic
is generally of lamin increase
to differentiate an increased
EC, embryonal RA, retinoic acid;
B remains when along
the
expression
unchanged, HL-60
human
macrophage of
lamins
carcinoma; GAPDH, SDS sodium dodecyl
0006-291 X/92 $4.00 Cop>lright 0 1992 by Academic Prex. Irrc. Ail rights of reproduction in atty form rerervd.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUillCATIONS
Vol. 189, No. 3, 1992 A and C accompanies The EC cell
differentiation
lines
expression
of lamins
A and C also
display
expressed
only
Moreover,
increased
changes of
lamins
A
It
has
C is
with
been
demonstrated
level
This
(9).
of
these
begun
not
(8).
only
on
and
Lamins
development,
in
mouse
v-ras"-induced
Thus,
it
cell
type,
being
embryos
(3).
phenotypic
seems that but
expression also
on
the
cells. that
correlated in the
quantitative
in HL60 cells synthesis
dependent
(3-5).
embryonic
has
(11).
cells state
differentiation
during
cells
U937 tumor
undifferentiated
A and C accompany
cancer
of the
changes
following
differentiation of lamins
F9 cells
Moreover,
correlate
expression
tissue
state
undifferentiated (6).
differential
human lung
and
B in their
occurs
levels
in NCI-H249
lamin
A and C only
after
differentiated
of human monoblastoid
P19 and F9 express
with
rate
lack
of
the absence
of synthesis
variations
prompted is
lamins
the
regulated
A
and
C
of the corresponding
of lamins
in the abundance
correlation
polypeptides
the
mRNAs
A and C were shown to
of their
authors
primarily
in
respective
to conclude at
the
mRNAs that
the
transcriptional
(9).
In this cells
study,
(12,13).
muscle
These
in response
RA as aggregates treated
with
do
express
not
we have analyzed cells
have
to DMSO (13)), (13),
or
lamins
into
into
RA as monolayers
the
In their
C;
however,
We show that
regulation
(4).
C occurs
at a post-transcriptional
to
neurons
(14).
A and
expression
potential
fibroblast-
differentiation mainly
the
of
lamins
differentiate
and glial
cells
and smooth they
state,
these
these
expression
lamins of lamins
P19
cardiac
when treated cells
acquire
of the
into
muscle-type
undifferentiated
level
A and C in
with when cells during A and
in P19 cells.
MATERIALS AND METHODS Cell
culture and materials P19 cells were cultured and differentiated by treatment with RA on monolayers or on with DMSO as described aggregates or in (15). Immunofluorescence and immunoblotting detection of lamins A and C in P19 cells were performed as previously described for F9 cells (5,16). RA was purchased in 95% ethanol (IBI) and from Kodak; a stock solution of 10e3 M was prepared a-[32P]-dCTP (800 Ci/mmol) was purchased kept at -20°C for no more than a week. from ICN and a-[32P]-GTP (3000 Ci/mmol) from Amersham Corp. (Arlington Heights, vanadyl-ribonucleoside complex was purchased from Bethesda Research IL); Laboratories. RNA blot analysis Isolation of cytoplasmic RNA was as in (17). Poly(A)' RNA was fractionated on 1.2% agarose-2.2 M formaldehyde gel (18), with 10 m M phosphate (pH 6.5) as running buffer. Hybridization was carried out at 42°C overnight. The probe was a plasmid bearing the entire coding sequence of murine lamin C (19) kindly provided by Dr. D. Werner (German Cancer Research Center, Heidelberg, Germany). After stringent washes, the blot was exposed to Kodak XRP film. Nuclear
run-on transcriotion assavs Nuclei were isolated as described in (17). assays were carried out essentially as described
1640
Nuclear in (20),
run-on transcription with modifications.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 189, No. 3, 1992
Nuclei (100 pi/assay), isolated as described above, were allowed to thaw on ice and mixed with 100 ~1 of reaction buffer (10 m M Tris pH 8.0, 5 m M t$Clz, 300 m M KCl, 0.5 m M each of ATP, CTP and UTP, 10 /&l GTP, and 200 &i of a-[ PI-GTP (300 Ci/mmol, Amersham)). Reaction was incubated at 27°C for 30 min, at the end of which each reaction received 200 ~1 of 10 m M Tris pH 8.0, 1 m M EDTA, 200 m M vanadyl-ribonucleoside complexes, 25 pg/ml tRNA, 10 mg/ml RNase-free DNase (BRL), was passed a few times through a syringe (18 l/2 gauge), and incubated 30 min at room temperature, followed by proteinase K (75 pg) treatment in the presence of The labeled RNA was purified by 2 phenol-chloroform1% SDS, at 37°C for 1 h. isoamyl alcohol extractions, chloroform extraction and ethanol precipitation. RNA was resuspended in 100 ~1 of sterile Hz0 and passed through a Sephadex G-50 column. Radioactivity in each reaction was determined by Cerenkov counting. In each filter was hybridized with the same number of cpm a given experiment? (usually 0.5 - 1x10 cpm) of labeled RNA. To prepare filters for hybridization, 5,ug of plasmid (pGEM), or plasmid containing cDNA for murine lamin C, described above, or for actin or GAPDH genes were denatured by incubation with 0.2 M sodium hydroxide for 20 min at room temperature, and neutralized with 3 volumes of 50 m M Tris pH 8.0, 1 M NaCl, 50 m M EDTA and 15 volumes of 333 m M Tris pH 6.8. DNAs were spotted onto Biotrans nylon membrane (ICN) using a Biorad slot blot apparatus, UV cross-linked for 2 min and baked at 80°C for 90 min. Filters were pre-hybridized for 6 h at 42°C in 50% deionized formamide, 5xSSPE (1xSSPE = 0.15 M NaCl, 10 m M Na PO,H,O, 1 m M EDTA), 5 x Denhardt's solution (21), 100 ,ug/ml yeast tRNA, 1 m M NaPP6,, 0.1% SDS. At the end of 6 h, buffer was replaced with freshly-prepared buffer and labeled RNA was added. Hybridization was carried out at 42°C for 40-48 h. Filters were washed with 2xSSPE, 0.1% SDS 2x5 min at room temperature, 3x30 min at 55"C, followed by a wash with 0.1 SSPE, 0.1% SDS for 30 min at 65°C. Filters were then treated with lOpg/ml RNase A and 15,000 u/ml of RNase Tl in 2xSSC, at 37°C for 30 min, followed by a final rinse at 37°C in 2xSSC for 15 min. Filters were exposed to Kodak XAR films for a maximum of 2 weeks. RESULTS AND DISCUSSION The kinetics during
differentiation
P19 cells treatment
(Fig.
treatment
RA for
These
8).
materials
RNAs were
the entire
coding
hybridize
with
extensive
portion
Fig. to
mRNAs for of their
at
24 h of
differentiated
along
murine
both
lamins
(lane
6),
RA treatment
abundant
those phorbol
for esters
cultures
of
3),
24 (lane
P19 cells
RA on aggregates
lamin
C (19).
present
at
Levels
1).
(lane than
low
of both
4)
probe
(lanes
6-8).
those
lamin
A.
lamin
A
(9). 1641
when In
HL60
addition,
lamins
share
an
Indeed,
kb corresponding
detectable
levels
gradually
a maximum
pathways for
therefore
(22,23).
and 2.1
species
under
containing
should
two
but
and reach
7) or DMSO (lane
plasmid
sequences 3.0
following
as described
these
and coding
of approximately
following
cells (lane
This
A and C since
from
4) and 72 h (lane
differentiated
and hybridization,
each of the three
more
than
fully
are
C mRNAs were more abundant with
from
5' non-coding
lamin
differentiate
8 (lane
determined
RNA was isolated
used was a nick-translated
for
A and C, respectively, PI9 cells
2),
by blotting
two mRNA species
undifferentiated starting
(lane
The probe
sequence
1 shows that lamins
extracted
analyzed
and methods.
poly(A)'
monolayer
1, lane
RA on monolayers
A and C mRNAs were first
Cytoplasmic
1) and from
2 (Fig.
RNA was also
with
of lamins
of P19 cells. 1, lane
with
Poly(A)'
5).
of accumulation
in
At all
in
increase cells time
full,y points,
Lamin C RNAs were also cells the
were time
induced
course
of
to RNA
Vol.
189,
No.
BIOCHEMICAL
3, 1992
AND BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RA
,,,,“2~EH Lamin
A w
Lamin
C *
kb
RA
\
I
_ 7.5 - 4.4 _ 2.4
12
0
48
72 h
_ 1.4
pGEM w Actin w GAPDH w
01
0
2
GAPDH w
Lamin A/C
w
Fiqure 1. Time course of accumulation of lamins A and C mRNAs in differentiatinq P19 cells. Poly(A)' RNA was isolated from undifferentiated P19 cells (lane 1) and from monolayer cultures of P19 cells treated with RA for 2 (lane 2), 8 (lane 3), 24 (lane 4) and 72 h (lane 5), or 7 days (lane 6). Poly(A)' RNA was also extracted from fully differentiated cells that were treated as aggregates with RA (lane 7), or with DMSO for 10 days (lane 8). Each lane received 5 pg of poly(A)' RNA. RNAs were fractionated on an agarose-formaldehyde transferred to a nylon membrane and hybridized with a nick-translated gel, plasmid containing the entire coding sequence for murine lamin C (19). The membrane was stripped of probe and rehybridized with a nick-translated plasmid containing a GAPDH cDNA insert. Run-on transcriotion assav with nuclei isolated at different staqes Fiqure 2. of RA-induced differentiation of P19 cells. Nuclei were isolated from cells used in the experiment described in Fig. 1: undifferentiated P19 cells, monolayer cultures of P19 cells treated with RA for 12, 48, 72 h, as indicated above each lane. Run-on transcription assays were as described under materials and methods, labeled RNAs were hybridized for 48 h with plasmid containing cDNA inserts (5 kg) immobilized on nylon membranes. Positive (GAPDH and actin) and negative (pGEM) controls are shown.
accumulation
in
A and C accumulation detected
total
in F9 cells,
24 h after Expression
loaded
P19 cells
differentiating
the
onset
of the
in each lane accumulation
in which
increased
of differentiation
the time
to
amounts
Taking
1).
lamins
into
course
of both
of lamin
lamins
were
(5).
GAPDH gene was used as a control
(Fig. of
is similar
account
for
the
the
levels
A and C mRNAs seems more
amounts
of RNA
of GAPDH RNA, the
important
at later
times
of RA treatment. The
presence
undifferentiated number
of
A and C.
of P19 cells
spontaneously Both
not
differentiated
of
mRNAs
be attributed cells
to
P19 cell
confirmed
lamins the
expressing
and immunoblotting (22)
for
the
cultures
(data
sharp
increase
complete not
presence
a high
with
A level
and of
C
in
a small
of
lamins
a highly
specific
lamin
absence
of lamins
A and
shown),
as previously
shown in
(3,4).
To determine was a consequence nuclei
could
antibody
C in undifferentiated studies
amount
small
immunofluorescence
A and C monoclonal other
a
were isolated
whether of
the
transcriptional from
the cells
activation used in the 1642
observed of
in
the
RNA blotting
lamins
lamin
A and C mRNAs
A and C gene(s), experiment
(Fig.
1)
Vol.
189,
No.
and subjected counts
BIOCHEMICAL
3, 1992
to
from
run-on
each
recombinant
transcription
reaction)
plasmids of lamin
cDNA insert
used contains exists
splicing
of
the
transcription
the
that
lamins
of these
pattern
starting
C mRNAs may
unchanged the
the
during for
of lamin C.
in
the
first
lamins
C since
the
However,
the
by
Fig.
2,
alternate
the
three
pattern
of the
experiment,
be produced
transcription
onset
this
lamin
As shown
of mRNAs specific
at 24 h after
that of
RNAs (equal
insert-containing In
sequence
(22,23,24).
Therefore,
of accumulation
increase
A and
denatured from
COMMUNlCATiONS
labeled
filters.
coding
RNAs remains
process.
to
nylon
be distinguished entire
RESEARCH
Extracted
hybridized on
same transcript
differentiation the
were
A cannot
BIOPHYSICAL
assays.
immobilized
transcription possibility
AND
rate
days of the
does not
A and C which
differentiation
of
reflect showed an
process
(Fig.
1). Taken C is
not
cells.
together,
regulated Instead,
responsible
these
for
is
in
gene expression
known that the
RA-induced normally
which
degrade
processing,
to
lamins or
progresses.
lamins is
result
RNA and protein
studies
altered
of
synthesized
a newly
either
A and
C RNAs.
transport
mechanism
is
of
changes
of
proteins.
stability
24 h after of these
a polypeptide
RNAs,
that
polypeptide
lamins
time.
by dramatic
would
that
Alternatively,
experiments
was not addressed
on the
confers
a more
rapid
A and C RNAs may occur
are
in particular
apparent cell
of lamins
A and C was mainly
the
of regulation
concerning
under
as
way to discriminate
directly
level
in these
studies
lines of the
since
Therefore,
our results
are the first
in
expression
of
A and C is
lamins
correlation
between
levels
that
regulation
to conclude
transcriptional
was performed. transcriptional
P19
any given
expression
of an increased of
A and of
A and C mRNAs starting
synthesis
had relied
synthesized
of the expression
P19 cells,
A and C at
accompanied
in
of lamins
Further
of lamins
differentiation
possibilities.
Previous
question
or that
polyadenylation these
process
the
expression
during
a post-transcriptional of
is the result in
them,
stability
that
ultimately
accumulation
that
level
synthesis
the differentiation
modulation
differentiation
demonstrate
suggest the
differentiation
a greater
between
results
increased
reflecting
results
transcriptional
regulating
It
Perhaps
these at the
(9).
synthesis
no run-on
of
lamins
transcriptional
demonstration mostly
However,
regulated
of the
A and C assay
that, at
at least a post-
level. ACKNOWLEDGMENTS
We thank Dr. F. McKeon for a generous gift of anti-lamin AC monoclonal antibody, Dr. D. Werner for a gift of the murine lamin C plasmid, P. Hince for technical assistance, R. Duclos for photography and C. Nault for typing the manuscript. J.L. was supported by a fellowship from the Cancer Research Society, J.F.C. by studentships from the Quebec Ministry of Education and Universite de Montreal, and Y.R. by a chercheur-boursier award from the Fonds de la recherche en Sante du Quebec. This work was supported by grants from the Medical Research Council of Canada.
1643
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189,
No.
BIOCHEMICAL
3, 1992
AND BIOPHYSICAL
RESEARCH
COMMUNICATIONS
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