[CANCER RESEARCH 45, 6051-6057,
December 1985]
Changes in Nuclear Proteins on Transformation of Rat Epithelial Thyroid Cells
by a Murine Sarcoma Retrovirus
Vincenzo Giancotti,1 Maria Teresa Berlingieri,2 Pier Paolo DiFiore,2 Alfredo Fusco,2 Giancarlo Vecchio,2 and
Colyn Crane-Robinson
Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Università di Trieste, Trieste 34127, Italy [V. G.]; Centro di Endocrinologia ed Oncologia Sperimentale
del CNR, Dipartimento di Biologia e Patologia Cellulare e Molecolare, II Facoltà di Medicina e Chirurgia dell'Università di Napoli, via S. Pansini 5, Napoli 80131, Italy [M.
T. B., P. P. D., A. F., G. V.]; and Biophysics Laboratories, Portsmouth Polytechnic, St. Michael's Building, White Swan Road, Portsmouth, P01 2DT, Hants, United Kingdom
[C. C-R.]
ABSTRACT
Two-dimensional electrophoresis has been used to document
changes in nuclear proteins following viral transformation of an
epithelial cell line exhibiting differentiation markers. After trans
formation, these markers are lost, and the cells become tumorigenie and capable of growth in soft agar. A sharp rise in the
phosphorylation of histones H1, H2A, and ubiquitinated H2A is
seen on transformation, together with the appearance of three
phosphorylated proteins that are extractable by perchloric acid
and appear related to high mobility group Protein 14, a constit
uent of active chromatin. Since comparison is made between
normal and transformed cells that are each grown to confluence
and since there is little difference between their observed growth
rates, the changes seen represent intrinsic differences between
the cell lines and are thus a direct reflection of the process of
transformation.
INTRODUCTION
It is well established that cellular transformation induced by
the oncogenic retrovirus Kirsten murine sarcoma virus is due to
the virally encoded protein v-ras (1). The detailed mechanism by
which this protein brings about the transformed state is not clear,
but it is likely that altered transcription of a number of cellular
genes, e.g., those of growth factors, is induced as a secondary
effect of the presence of the transforming protein. Although a
number of changes in the level of certain unidentified nuclear
nonhistone proteins have been observed (2) and a stimulation of
protein phosphorylation has been noted (3-5) in transformed
cells, few specific observations have been made on the state of
the histones and HMG3 proteins except for the observation of
increased phosphorylation of histone H1 (6). We have therefore
compared the state of basic nuclear proteins in a virally trans
formed cell line with that in its normal, untransformed state. The
cells chosen were Fischer rat thyroid epithelial cells which have
been transformed by Kirsten murine sarcoma virus into a state
in which (a) their characteristic differentiation markers (growth
dependence on thyrotropin, iodide uptake, and synthesis/secre
tion of thyroglobulin) are lost, and (b) they are able to grow in
soft agar and are tumorigenic when transplanted into syngenic
animals. These cell lines are more fully described in Refs. 7 and
Received 3/25/85; revised 7/15/85; accepted 8/1/85.
1 Recipient of support from the Ministero della Istruzione, the CNR of Italy, and
the University of Trieste. To whom requests for reprints should be addressed.
2 Recipient of support from the Progetto Finalizzato Virus of the CNR of Italy.
3 The abbreviations used are: HMG, high-mobility group protein; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; TEMED, tetramethyl ethylene diamine; RSB, 10 DIM NaCI/10 mm Tris-HCI/3 HIM MgCI2 (pH 7.4).
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RESEARCH
8. A significant feature of this system is that the cells are epithelial
rather than the more usual fibroblasts and have clear differentia
tion markers. They are thus a better model for human carcino
mas, some of which have been shown to involve the activation
of the cellular counterpart (c-ras)Ki of the Kirsten murine sarcoma
viral transforming gene (9). The comparison between normal and
transformed cells has shown a marked increase in phosphory
lation of histones H1, H2A, and its ubiquitinated form uH2A in
transformed cells and the appearance of 3 phosphorylated pro
teins not observed in normal cells.
MATERIALS AND METHODS
Cells and Viruses. Fischer rat thyroid differentiated cell clones, de
rived from Fischer rat thyroid glands as described by Ambesi-lmpiombata
ef al. (10), are designated here AN. They were grown in a modified Ham's
F-12 medium containing 5% calf serum (Grand Island Biological Co.,
Grand Island, NY) plus 6 growth factors (insulin, hydrocortisol, transferrin,
thyrotropin, somatostatin,
and the tripepetide glycyl-histidyl-lysine).
Transformed cells, designated AT, were obtained by plating AN cells at
3 x 105/dish and then infecting the next day with 1.0 ml of undiluted
fresh supernatants
from Kirsten murine sarcoma virus-infected
NRK
58967 cells in the presence of polybrene. Both normal and transformed
cells were grown to confluence and left for more than 24 h before
harvesting. In experiments using 32P label, the culture medium was
replaced by one lacking phosphate 6 h before harvesting and after a
further 2 h replacing the medium with one including 40 nC\ of [MP]phosphate per ml. Cell harvesting thus followed after 4 h in radioactive
medium.
Preparation of Nuclei. Cells were detached with a solution of phos
phate-buffered saline (137 HIM NaCI/2.7 mw KCI/1.47 mw KH2PO4/8.1
mM Na2HPO4, pH 6.6) containing 3 mg of trypsin per ml. After 5 min in
this solution, cells were collected by low speed centrifugation at room
temperature. The cellular pellet was resuspended in hypotonie RSB at
10-40 x 106 cells/ml and left on ice for 10 min. Nonidet P-40 (0.5%) was
then added, and the suspension was left for a further 5-10 min on ice.
After centrifugation at 1500 x g for 10 min at 0°C, the process was
repeated on the pelleted material using the same RSB solution plus
0.2% Nonidet P-40. Nuclei were finally washed twice with RSB solution
without Nonidet. The nuclear pellet was preserved at -80°C when not
extracted immediately.
Protein Extraction. Direct extraction of nuclei was with 0.20 M H2SO4
containing 1 ITIM butyric acid and 0.2 mM PMSF for 2 h on ice with
stirring followed by centrifugation for 15 min at 60,000 x g. The super
natant was dialyzed against 1 mw HCI/1 mM butyric acid/0.2 mM PMSF
using a membrane with a molecular weight cut-off of 3500, and the
protein was recovered by lyophilization. Selective extraction of histone
H1 and HMG proteins with 4% (w/v) perchloric acid was 3 times with 1
ml for the nuclei derived from 50 x 106 cells. After centrifugation at 3000
rpm for 2 min at room temperature, the supernatant was made 0.3 M in
HCI, and then 9 volumes of acetone were added. The solution was left
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PROTEIN CHANGES
in the freezer at -15°C overnight. The precipitate
ON RETROVIRAL TRANSFORMATION
was recovered by
repetitive centrifugaron in a single Eppendorf vial, washed twice with
acetone, and vacuum dried at room temperature. Acid-soluble proteins
remaining in the pellet after perchloric acid treatment were extracted by
addition of 0.2 M H2SO4/1 mw butyric acid/0.2 mw PMSF, as described
above for whole nuclei.
Electrophoresis. Acetic acid/urea gels were in 15% polyacrylamide/
0.2% bisacrylamide/0.2% ammonium persulfate/0.5% (v/v) TEMED in
0.9 M acetic acid/4 M urea. The stacking gel was 7.5% polyacrylamide/
0.2% bisacrylamide/0.2%
ammonium persulfate/1 % TEMED/0.9 M
acetic acid/4 M urea. The thickness was 1 mm, and preelectrophoresis
was overnight at room temperature with 30 ¡Aper slot of 1 M cysteamine/
15% sucrose in the above acetic acid/urea solvent, at a constant 7 V/
cm. Samples were dissolved in the gel solvent plus 1% /3-mercaptoethanol and 15% sucrose. The upper and lower reservoirs contained only
0.9 M acetic acid. Samples were accumulated at the Interface using a
constant 50 V for 2 h and separated at a constant 210 V for 30-35 h at
room temperature. Staining was with 0.2% Coomassie R250 in methanol
acetic acid/water (5/4/1) and destaining with ethanol/acetic acid/water
(5/13.4/1.6). When the gel was to be used for a second dimension, 5%
glycerol was added to the destain solution. For autoradiography, gels
were washed for 2 h in ethanol/water (1/3) before vacuum drying at
80°C. Second dimension gels (1.5-mm thickness) were in 15% polyacrylamide/0.20% bisacrylamide/0.075% ammonium persulfate/0.075% (v/
v) TEMED dissolved in 0.75 M Tris-HCI, pH 8.5, 0.1% SDS. Treatment
of first dimension polyacrylamide strips for second dimension analysis
was by soaking for 30 min in 0.75 M Tris-HCI, pH 8.8, 1% SDS before
setting in fresh 15% polyacrylamide. Separation was at a constant 5 V/
cm for 14 h. The Coomassie blue from the first dimension was used to
indicate the migration front. Staining, destaining, and drying were as
sie-stained gel of the sulfuric acid-extracted proteins and its
autoradiograph. Intense phosphorylation of the H1 region of the
gel is apparent for transformed cells (AT) as compared to the
normal cells. In addition there is phosphorylation in the H2A/H2B
region of the gel and for the ubiquitinated form of H2A (uH2A) in
the transformed cells but not in the normal. In order to better
define the level of H1 phosphorylation, the distinct H1 bands
(Nos. 1 to 5; see Fig. 1) were separately cut from the dried
Coomassie-stained gel of AT in Fig. 3 and individually autoradiographed. The result is shown as the inset to Fig 3, from which
it is clear that Band 1 is unphosphorylated H1, whereas the other
bands contain phosphorylated H1 species. In particular, the
minor Band 5, which is totally absent from normal cells, is seen
to be strongly phosphorylated from the high ratio of ^P to
Coomassie stain.
Bands 2 to 4 also contain phosphorylated species. The same
experiment was also carried out with the 2 Coomassie-stained
H1 bands from AN of Fig. 2. Only the upper band, corresponding
to subspecies 2, contained label (data not shown). So as to
examine in more detail the phosphorylation and other changes
of basic nuclear proteins on transformation, 2-dimensional gels
were obtained using 0.1% SDS for the second dimension. Fig.
4 compares Coomassie-stained and autoradiographed gels of
the 32P-labeled preparation seen in Fig. 3. The region covered in
the 2-dimensional gel is from H1 to H4. From the Coomassiestained panels, it can be seen that there is no obvious change in
the overall core histone pattern, but there is an apparent reduc
tion in the amount of H1 °in the transformed cells (as also seen
described for acid/urea gels.
Autoradiography. Cromex cassettes were used at -80°C with XAR-
in Fig. 2) and no sign of its phosphorylation. Much greater
differences are, however, seen in the autoradiograph. The in
5 Kodak X-ray film.
creased phosphorylation of H1 has already been described.
Overlapping the Cooomassie-stained gel and the autoradiograph
of AT (Fig. 4) shows that the major core histone label runs slightly
RESULTS
ahead of H2B in the second dimension and must therefore be
Fig. 1a shows a Coomassie-stained acetic acid/urea gel of due to phosphorylated H2A. Comparison of the Coomassie stain
and autoradiography of H2A in Fig. 3 shows that the label runs
sulfuric acid-extracted total nuclear protein from normal (AN) and
slightly behind the bulk of the Coomassie stain; i.e., only a small
virally transformed (AT) cells. Fig. 10 gives an enlargement of
proportion of the H2A is phosphorylated. This phosphorylation
the histone region of Fig. 1a. Fig. 1c is a similar gel obtained
of H2A in AT correlates with the phosphorylation of ubiquitinated
after a longer time of electrophoresis to give a greater expansion
H2A (uH2A) seen in Fig. 3.
of the H1 region. Histones were identified by comparison with
This protein (Component P6) from Fig. 4 does not appear to
calf thymus standards. In this gel system, proteins modified by
alter
its overall level on transformation but clearly becomes
acetylation and phosphorylation have a retarded mobility. The
phosphorylated,
since there is no sign of radioactivity in P6 for
most obvious difference between AN and AT is the presence of
the normal cells. The second dimension suggests that P6 could
H1 species of reduced mobility in AT (Bands 1 to 5). To check
be an H3 subfraction that becomes phosphorylated, since P6 is
that all bands in the H1 region are indeed H1 species, nuclei
precisely in line with the set of acetylated H3 subfractions
were treated with 5% perchloric acid to selectively extract H1,
and the proteins then separated on an acid/urea gel, as in Fig. indicated by the triple arrows. Such a suggestion can only be
substantiated, however, by purification and sequencing.
1. After electrophoretic transfer to nitrocellulose paper in dupli
Components labeled A-D in the autoradiographs of Fig. 4 are
cate, one copy was stained with amido black, and the second
present
at low levels, since they are not visible in the Coomassie
was treated with anti-H1 serum (11). Fig. 2 shows the results,
panels.
Of
these, A appears less phosphorylated in the trans
which demonstrate that all the bands in the H1 region, in partic
formed cells, and B to D are more phosphorylated, but without
ular that designated No. 5, are recognized by the antiserum and
knowing the relative absolute levels of these proteins in AN and
can therefore be regarded as H1 species. The faster migrating
AT, changes in their degree of phosphorylation cannot be esti
band that is also recognized by the H1 antiserum is almost
certainly due to histone H1°,since we have separately shown
mated from Fig. 4. Component C appears to consist of a single
spot with a horizontal tail to the left-hand side. Although it might
that this antiserum recognizes H1°from Chinese hamster ovary
be due to degradation, heterogeneity in the first but not the
and V79 cells. The multiplicity of H1 bands could be due to either
phosphorylation or new subfractions of H1, or to both. 32P- second dimension is not readily explained.
A particular property of histone H1 and the HMG proteins is
labeled phosphate was therefore added to cell cultures as de
scribed in "Materials and Methods." Fig. 3 shows a Coomas
their solubility in 5% perchloric acid. In order to examine the
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PROTEIN CHANGES
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HMG components, nuclei from 32P-labeled cell cultures (AN and
AT) were extracted first with 5% perchloric acid and then with
0.2 M HjSCv The 2 extracts were separately run in a first acid/
urea dimension and then together in a second SDS dimension.
After Coomassie staining (upper panels of Fig. 5), the gels were
autoradiographed (tower panels). In the first dimension of the
perchloric extract of both AN and AT, strong bands due to H1
and H1 °can be seen and also weaker bands from HMG 14 and
17. In the autoradiographs of the second dimension, it can be
seen that in this experiment H1 from AN cells is considerably
phosphorylated in comparison to that seen in Fig. 3. This is due
to a much higher level of isotope incorporation in the experiment
of Fig. 5 as compared to that of Fig. 3, resulting from the use of
a higher concentration of [^PJphosphate. A check was made
that the distribution of label among the H1 subfractions of AN
and AT in the experiment of Fig. 5 was the same as that shown
in Fig. 3 for the earlier experiment, i.e., 2 bands for AN (only the
upper labeled) and 5 bands for AT (of which the upper 4 were
labeled). Fig. 5 shows the presence of 2 H1 °subfractions, one
of which (the faster migrating in SDS) is labeled.
The experiment of Fig. 5 with a high level of isotope incorpo
ration gave a clearer demonstration of the higher level of phos
phorylated Protein A in the untransformed cells relative to the
transformed. Since, however, the protein A is not visible in the
Coomassie panels of Fig. 5, it is not possible to be certain if, on
transformation, less of Protein A is made, or Protein A is dephosphorylated. The complete absence of P6 and H2A phosphorylation in AN, relative to AT, is particularly evident in Fig. 5.
Selective extraction with 5% perchloric acid is valuable to ob
serve the low-molecular-weight HMG Proteins 14 and 17. These
were identified in the first dimension acid/urea (top of Fig. 5) by
comparison with calf thymus standards. In the Coomassiestained second dimension of Fig. 5, HMG 14 and 17 are present
in approximately equal amounts in both normal and transformed
cells, but from the autoradiographs, it is seen that only HMG 14
is phosphorylated, not HMG 17. The position of phosphorylated
HMG 14 in the autoradiographs is slightly to the right of the
maximum of Coomassie stain, i.e., migrates more slowly in the
first dimension, as expected for a reduced positive charge. There
is no clear difference in the level of phosphorylation of HMG 14
in the transformed and normal cells. The Coomassie-stained gel
of AT in Fig. 5 shows the presence of Proteins C to E in the
perchloric acid extracts of the transformed cells, while they are
not seen in the gel of the untransformed cells. The autoradiograph of AT in Fig. 5 shows all these 3 proteins to be phos
phorylated. The radioactivity peak of Proteins D and E coincides
with that of Coomassie staining, indicating their complete phos
phorylation. Component C is somewhat spread out in both the
Coomassie-stained and autoradiographed panels of AT in Fig 5.
Component C could have multiple phosphorylated states, all of
which run identically in SDS. However, we have not been able
to observe multiple peaks of radioactivity in short-exposure
autoradiographs. It should be noted in passing that the compo
nents labeled F1,2 in the AN extract of Fig. 5 are in fact low
amounts of H2A and H2B removed during the 3x extractions
with perchloric acid. Finally, note should be made of the low but
significant level of H4 phosphorylation observed in the trans
formed cells (see Fig. 5 autoradiograph) but not in the normal
cells, despite their relatively high level of isotope incorporation.
Careful inspection of Fig. 4 also shows the presence of phos
CANCER
RESEARCH
phorylated H4 in AT. In Fig. 4, the streak of radioactivity in H4
mirrors the Coomassie stain of the H4 species but displaced to
lower mobility, implying a low level of phosphorylation of all H4
species present, acetylated and nonacetylated.
DISCUSSION
In comparing proteins extracted from the nuclei of normal
thyroid cells (AN) with those of the virally transformed cells (AT),
certain points concerning their growth must be stressed. First,
both AN and AT grow in culture at approximately the same rate
(doubling time, 24-36 h); I.e., differences are not due to large
variations in their relative rates of proliferation. Second, both
types of cell are grown to monolayer confluence at similar
densities and then left for at least 24 h before harvesting. This
ensures that the vast majority of cells are in the static quiescent
state Go when proteins are extracted. An advantage of using
this system is thus that effects resulting from proliferative growth
should be excluded, so that the observations made reflect intrin
sic differences in the nuclear proteins of AN and AT. Despite the
similarities between AN and AT, it must be remembered that,
although AN is a cell line capable of indefinite growth, it is still
dependent on thyrotropin and maintains characteristics of the
differentiated state.
There have been several reports of increased kinase activity
in transformed cells (3-5). The Ha-ras M, 21,000 protein has
been shown to have GTP hydrolytic activity (12), and the equiv
alent protein from Kirsten murine sarcoma virus has been shown
to bind GTP and to have autophosphorylating activity (13). The
transformed cells studied here show elevated levels of phos
phorylation in certain nuclear proteins, e.g., H2A and Protein P6.
There is also an increase of phosphorylated proteins in the
transformed cells, e.g., Proteins C to E. Increase of phosphory
lation may not be universal in this system, however, since
phosphorylated Protein A is less abundant in the transformed
cells.
Increased phosphorylation of H1 in AT is particularly evident
in Fig. 3. This is not accompanied by phosphorylation of H3, as
found for mitotic H1 phosphorylation (14), a result suggesting a
distinction between these 2 types of H1 phosphorylation. The
autoradiography of Fig. 3 shows a very high level of label
incorporation into the weak Band 5 that is not seen in AN at all.
This suggests that a high degree of modification of at least one
subfraction of H1. More detailed experiments are needed to
distinguish between the phosphorylation induced in the various
H1 subfractions.
The second striking difference of histone phosphorylation be
tween AT and AN is in H2A which is strongly phosphorylated in
AT but not in AN. This core histone is typically found to be
partially phosphorylated throughout the cycle of growing cells
(15), and the absence of label in AN is thus unexpected. The
phosphorylation of the ubiquitinated form of H2A (uH2A) faithfully
mirrors that of the parent protein, suggesting that ubiquitination
occurs independently of the state of phosphorylation.
Perchloric acid extraction of the transformed cells shows the
presence of 3 new phosphorylated proteins, C to E, not observed
in AN, but apparently present in significant quantities in AT (see
Fig. 5). Judging from their mobilities in both dimensions, these
may be members of a recently described group of HMG proteins
similar to HMG 14 and 17 but which appear only in proliferating
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PROTEIN CHANGES
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cells. Thus HeLa cells have been shown to contain, in addition
to HMG 14 and 17, two proteins (HMG I and M) with mobilities
just slower than HMG 14 on acid/urea gels (16). Similarly, rat
thymus from young animals has a protein HMG I migrating behind
HMG 14, while rat fibroblasts transformed with avian sarcoma
virus have 2 additional HMG 14/17-like proteins, HMG I and I'
3.
4.
(17). Proteins C and D in Kirstein virus-transformed cells probably
correspond to HMG I and I'. The Protein E could be another
5.
variant of HMG I or could be a variant of HMG 14, since this
protein is known to have multiple forms. The appearance of
Phosphoproteins C to E on transformation suggests a common
function of the HMG I and HMG 14 proteins distinct from that of
HMG 17, which is not phosphorylated in these cells. Proteins C
to E might also be related to Proteins PS1 to 3 that have been
observed in perchloric acid extracts of calf thyroid tissue (18).
Protein PS2, in particular, is strongly phosphorylated. These
results on the phosphorylation of HMG proteins agree with other
studies which have shown that, in interphase cells, HMG 14 and
I are the major HMGs phosphorylated (17-19). The HMG Pro
teins 14 and 17 have been implicated in the control of chromatin
structure in the region of active genes (20). The appearance of
Proteins C to E in the transformed cells might therefore be linked
to the induction or repression of cellular genes that lead in turn
to the ability for autonomous growth and the loss of differentiated
functions.
Finally we comment on Protein P6 which, while present in
approximately equal amounts in AN and AT, becomes phos
phorylated following transformation. This protein is designated
P6, since it is one of a group of 9 proteins observed in a range
of mammalian cells having mobilities in acid/urea gels slightly
less than that of H3. P6 is not a decomposition product of H1,
since it is not extracted by perchloric acid (see Fig. 5). From its
mobility on the 2-dimensional gels, it could be an unusual H3
subfraction, but its phosphorylation in AT when all the defined
H3 components remain unphosphorylated makes this unlikely.
Comparison with the 2-dimensional gels of Busch ef al. (21)
suggests that it may correspond to the protein designated A7
by them.
6.
ACKNOWLEDGMENTS
We are grateful to Dr. P. Symmons of the Institut fürGenetik, Dusseldorf, for
carrying out the tests with anti-H1 serum.
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AcOH/UREA
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AT
AN
AT
uH2A-
H1
uH2A-
H2B{
H2A1
H4
H4{
Fig. 1. Acetic acid/urea gels of sulfuric acid-extracted nuclear proteins from untransformed (AN) and transformed (AT) cell nuclei, a, a full length gel in which the
amounts of AN and AT loaded were equated for equal staining of the core histones; b, photographic enlargementof a showing the histone region of the gel; c, a second
gel of the same samples run for a longer time to achieve greater expansion of the histone H1 subfraction complexity.
AN AT
AN AT
«•5
«•5
»B
-
H1
amido black
antibody
Fig. 2. Perchloric acid extract of AN and AT nuclei separated on an acid/urea gel. The H1 region of the gel was electrophoretically transferred to nitrocellulose and
then stained with Amido black and also treated with rabbit anti-histone H1 antibody. Bound antibody was visualized by using phosphatase-conjugatedgoat anti-rabbit
antibody and then treating with 5-bromo-4-chloro-3-indolylphosphate (p-toluidine salt) which yields a sky blue precipitate (11).
CANCER
RESEARCH
VOL. 45 DECEMBER
1985
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AN
AT
I
4,
AN
AT
AcOH/UREA
n..-UH2A
uH2A-
H2A
COOMASSIE
Fig. 3. An acetic acid/urea gel of acid-extracted
AUTORADIOGRAPHY
nuclear proteins from AN and AT cells labeled for 2 h with [MP]phosphate. Loadings equated for equal Coomassie
staining of the core histories. The inset shows an autoradiograph of 4 separate slices cut from the H1 region of the gel. (No attempt was made to separate Bands 3 and
4 in this procedure.)
6—AcOH/UREA
HB
AT
\
H2A
H4
AN
\u
v
H3 P6
AT
H1
O
73
>
g
O
o
* «—A
D
l
\
TJ
I
Fig. 4. Second-dimension
autoradiographed.
B
C
l
H4
\
H2A
P6
SDS of the histone region of Fig. 3, as indicated therein. Normal (AN) and transformed (AT) cell nuclear proteins, Coomassie stained, and
6056
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1985 American Association for Cancer Research.
PROTEIN CHANGES
ON RETROVIRAL TRANSFORMATION
AcOH/UREA
HMG14
H4 ÕH2A H
l
r
l r
I
I
JH2B
HI-
<—AcOH/UREA
H4'H2AH3HMG17/HMG14
HI
ir
I
H\° I?1
II
If
I
CO'
HCIO,
Û
CO
HCIO4
I-
H1 'co
J,"
O
'O
ü
H2B0
H3
l
\ L
H4
\
__ \
H2A Ve
H3
HMG14
HMG14
\
\
2'A P6 HMG17
HMG17
E
e
HMG14
H4
\
HMG14
H2A
P6
AT
AN
Fig. 5. Two-dimensional electrophoresis of a "P-labeled preparation of normal (AN, panel a) and transformed (AT, panel b) nuclear proteins extracted first with 5%
perchloric acid (righi) and subsequently using 0.25 M sulfuric acid (left). The sulfuric and perchloric acid extracts were run in separate lanes of the same first-dimension
acid/urea gel. The sulfuric acid extract first dimension is limited to the core histone region of the gel. The perchloric acid extract first dimension covers a wider range
from above histone H1 to below HMG 17.
CANCER
RESEARCH
VOL. 45 DECEMBER
1985
6057
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Changes in Nuclear Proteins on Transformation of Rat Epithelial
Thyroid Cells by a Murine Sarcoma Retrovirus
Vincenzo Giancotti, Maria Teresa Berlingieri, Pier Paolo DiFiore, et al.
Cancer Res 1985;45:6051-6057.
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