[CANCERRESEARCH
39,507-518,February1979]
0008-5472/79/0039-0000$02.00
Two-Dimensional Gel Electrophoretic Comparison of Proteins of Nuclear
Fractions of Normal Liver and Novikoff Hepatoma1
Hiroshi Takami and Harris Busch
Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030
evidence for the presence of fetal proteins and other anti
gens in the tumors that are absent from nontumor tissues
The combination of two-dimensional isoelectnic focusing
(9, 41).
sodium dodecyl sulfate gel electrophoresis with successive
The improvements by O'Fammellet a!. (33, 34) and by
extraction of nuclei of Novikoft hepatoma and normal rat Peterson and McConkey (38) of the 2-dimensional gel
liver with (a) 0.075 M NaCI/0.025 M EDTA, (b) 10 mM Tnis, (C) system used by MacGillivray and Rickwood (29) and Liew et
0.35 M NaCI, (d) 0.6 M NaCI, and (e) 3 M NaCI/7 M urea al. (23, 28) for studies on nuclear proteins provide the
provides an improved approach to analysis of the number opportunity to advance rapidly the identification and char
and types of nuclear proteins. Each fraction contained 108 actenization of cellular proteins. The technique developed
to 200 spots, of which (a) some were present in all of the by Hirsch et a!. (20) in this laboratory provides highly
fractions, (b) some were present in more than one fraction,
reproducible separation of proteins on the basis of isoelec
and (C) others were uniquely found in one fraction. In the tnic point over the p1 range of 4.5 to 8.5 in the first
Novikoff hepatoma nuclei, 483 different polypeptides were dimension and by molecular weight on the SDS2 gels in the
found; 427 polypeptides were found in liver nuclei. The second dimension. Loading of the samples on the acid side
sensitivity of the method was such that spots containing 0.1 eliminated much of the streaking found when samples were
;Lg of protein were readily identified on these gels; such loaded on the alkaline side.
proteinsare presentinapproximately
500 to 600 copies/
The use of this procedure with minigels (20) has permitted
nucleus.
the identification of 5 cytoplasmic proteins present in rap
In the Novikoff hepatoma, 18 protein spots (designated
idly growing tumors but not in nontumom tissues on slowly
by molecular weight/pI) were found in the various nuclear
growing tumors. The method has been used recently to
fractions that were not found in the normal liver nuclei:
separate labeled in vitro-translated products from whole
170/6.0,
140/6.7,
140/6.8,
132/5.2,
97/8.45,
87/5.7,
84/6.3, mRNA or following preabsorption of mRNA on complemen
82/5.0,62/6.2,61/6.1,61/7.3,49/5.3,48/5.1,48/7.9,42/ tary DNA columns (22); several differences were found in
5.3, 38/7.3, 28/5.0, and 27/4.9. Ten of these proteins were mANA populations of regenerating rat liven and Novikoff
found in the 0.6 M NaCI extract. Earlier studies with other
hepatoma.
techniques showed several protein differences in this frac
The present study was designed to determine whether
tion between these tissues.
the increased resolution of the improved 2-dimensional gel
In normal liver nuclei, 12 spots were present in various
methods provides an improved separation of nuclear pro
nuclear fractions that were not found in the Novikoff hepa
teins, particularly those of high molecular weight in tumors
toma: 160/5.8,98/5.0,94/7.8,94/7.9,78/7.4,76/8.4,72/ and other tissues. To establish whether differences were
5.5, 65/5.2, 45/8.2, 41/5.6, 38/5.5, and 28/8.45 (molecular
present in their nuclear proteins, it first seemed desirable
weight/pI). These studies substantiate and extend earlier to study a system in which a maximum number of differ
studies from this laboratory which showed differences in ences might be found (20). For this purpose, Novikoff
nuclear nonhistone proteins in tumors and other tissues.
hepatoma ascites cells were used inasmuch as the cells can
They provide a basis for more definitive comparisons of be rapidly grown in sufficient quantities to permit precise
specific nuclear proteins in tumors of different growth rates chemical and sequence analysis of proteins and ANA as
and growing nontumonous tissues.
carried out in this laboratory for the histones, protein A24,
and nonhistone proteins, including phosphorylated pro
teins as well as low- and high-molecular-weight RNA spe
INTRODUCTION
cies. Fractionation of nuclear proteins in this study showed
Previous studies in this laboratory (2, 15, 42, 44) and the presence of 483 nuclear proteins (polypeptides) in
others (11, 27, 28) have reported that differences exist in Novikoff hepatoma cell nuclei and 427 in normal liver
the proteins of nuclei in tumors and other tissues. The nuclei. Although most proteins were common to both types
methods used included 2-dimensional gel electrophonesis
of nuclei, many differences were found.
of 32P-labeled and unlabeled proteins (15, 42-45) as well as
antigen-antibody reactions (6, 7). Analysis of the nuclear MATERIALSAND METHODS
and nucleolar antigens by immunodiffusion , electrophore
Animals and Tumor Cells. Novikoff hepatoma ascites
sis, and chromatographic techniques has expanded the
cells were obtained by drainage of the ascites fluid 6 days
after implantation into 200-g male albino rats (Holtzman
10893, P.1, awarded by the National Cancer Institute; Department of Health,
Education and Welfare, by the Pauline Sterne Wolff Memorial Foundation;
Co., Madison, Wis.). The ascites cells were filtered through
ABSTRACT
I These
studies
were
supported
by
Cancer
Research
Center
Grant
CA
by the Bristol-Myers Fund; and by a generous gift from Mrs. Jack Hutchins.
Received March 27, 1978; accepted October 23, 1978.
2 The abbreviation used is:SDS, sodium
dodecyl sulfate.
FEBRUARY1979
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507
H. Takami and H. Busch
cheesecloth and washed 3 times (30) with 0.13 M NaCI/
0.005 M KCl/0.008 M MgCI2. The normal livers were perfused
with this buffer and passed through a tissue press.
by the Bio-Rad assay method (Bio-Rad, Richmond, Calif.).
Approximately 60% of the sample was distributed through
the gel, and the remainder (basic proteins and cap proteins)
Isolationof Nuclei. Novikoffhepatomaascitescellsand either passed through the isoelectric focusing gel (2 to
normal liven pressates were homogenized in 0.5% Nonidet 21%) or remained at the top of the gel as a protein cap (32
P-40/0.01 M NaCI/1 .5 mM MgCI2, and 0.01 M Tmis-HCI, pH to 49%). The upper reservoir was filled with 0.01 M H3PO4
7.6 (31). The homogenate was centrifuged at 2300 x g for (anode), and the lower was filled with 0.02 M NaOH (cath
10 mm. The supernatant was used to prepare cytoplasmic
ode). Electmophomesiswas done at: (a) 30 mm at 50 V; (b) 30
protein as described earlier (20, 21). The crude nuclear mm at 100 V; (c) 200 V until the crystal violet tracking dye
pellet was resuspended by gentle homogenization in 2.2 M (10 @g)
ran off the alkaline end (approximately 1 hn); and (d)
sucrose/lO mM MgCl2 and centrifuged at 53,000 x g for 60 20 mm at 400 V. Under these conditions, these gels were
mm (41), followed by resuspension in 1 M sucrose/lO mM “at
equilibrium―with respect to the known markers stud
MgCI2 and centnifugation at 500 x g for 10 mm. The nuclei ied.6
thus obtained were morphologically satisfactory by light
The gels were removed and adapted for 3 mm in 0.05 M
Tnis-HCI, pH 7.5/1% SDS and frozen in a hexane/dry ice
microscopic analysis as described earlier (3). Phenylmeth
anesulfonyl fluoride (Sigma Chemical Co. , St. Louis, Mo.) bath for2 mm (26).The gelswere placedinthe running
was added fresh to all solutions used in this study in a final buffer (see below) containing 5% @3-mercaptoethanolfor 5
mm.
concentration of 0.5 m@i(4).
Nuclear Extraction. Nuclei were extractedsequentially The second-dimension slab SDS gel electrophoresis was
(40) with 0.075 M NaCI/0.025 M EDTA, pH 8.0, 10 mM Tnis carried out in a glass cell (83 x 83 x 1.2 mm). The slab gels
HCI, pH 8.0, and 0.35 and 0.6 M NaCI/lO mM Tnis, pH 8.0. were polymenized from a solution which contained 8%
The nuclei were gently resuspended by gentle homogeni
acrylamide, 0.3% N ,N'-methylenebisacrylamide, 0.1% SDS,
zation in 10 volumes of the above buffers, then placed on 0.2 M sodium acetate, 0.4 M Tnis-HCI, pH 7.5, 0.002 M EDTA,
ice for 20 mm with intermittent mixing, and centrifuged at 0.075% ammonium pensulfate, and 0.075% N ,N ,N' ,N'-tet
12,000 x g for 10 mm. The 0.075 M NaCl/0.025 M EDTA, pH namethylethylenediamine. The cylindrical first-dimension
8.0, and 10 mM Tnis-HCI, pH 8.0, extractions were pen gels were placed directly on top of the polymenized slab
formed 3 times; the other 2 extractions were performed
gels. The running buffer contained 0.4 M Tnis-HCI (pH 8.0),
twice.
0.1% SDS, 0.2 M sodium acetate, and 0.02 M EDTA. The
Following extraction with 0.6 M NaCI/lO mM Tnis-HCI, pH slab gels were run for 15 hr at 37.5 ma/slab gel.
The slab gels were removed and stained for 5 hr in 0.25%
8.0, the chromatin pellet was resuspended in 10 volumes of
3 M NaCl/7 M unea/lO mM Tnis-HCI, pH 8.0, and stirred Coomassie Brilliant Blue R in methanol/acetic acid/water
overnight. The dissociated chromatin was centrifuged at (5/1/5). Slab gels were destained in several changes of
350,000 x g for 24 hr (12).@
solution containing 10% acetic acid/10% isopropyl alcohol.
To measure the pH gradient, the isoelectnic focusing gel
Each extract was dialyzed for a minimum of 9 hr against
3 changes of 42 mM acetic acid (30/1 ,v/v), Iyophilized,4 and was cut into 5-mm sections and placed in individual tubes
with 100 @Iof H2O. On the following day, the pH was
subjected to 2-dimensional gel electrophonesis.
Two-Dimensional Gel Electrophoresis. The cylindrical measured with a pH meter.
Reproducibility of the Gels. Fig. 1, A and B , shows 2 gels
isoelectnic focusing gels (6.3 x 0.2 cm) were poured into
one-half of a 200-pi disposable glass micropet up to 0.8 cm prepared from the 2 different samples of nuclei purified by
the Nonidet-P40 method. As noted from the spot patterns,
from the top and overlayered with water. The gel solution
sizes, and densities, the results were highly reproducible.
contained 9 M unea/4% acrylamide/0.2% N,N'-methylene
bisacrylamide/2% ampholines, pH 3.5 to -‘-10
(LKB, Inc., Fig. 1C shows a gel prepared from a sample of Triton X
Rockville,
Md .), 0.02% ammonium
pensulfate/0.1 % 100-purified nuclei; the results were very similar to those in
Fig. 1, A and B. These results show that the Nonidet P-40
N ,N ,N' ,N'-tetramethylethylenediamine
(26). The original
samples (180 to 600 jig) were dissolved in 20 @d
of 9 M umea/ and Triton X-100 treatments did not produce marked
2% ampholine, pH 3.5 to 10, 2 mM dithiothneitol, and 10 @d changes in these patterns. Moreover, in Novikoff hepatoma
were loaded onto the acid side of the gel (22, 33, 34); and in normal and regenerating liven (18 hn), the percent
including losses from solubility and transfer, the actual load ages of proteins in the 0.075 M NaCl/0.025 M EDTA, pH 8.0,
on the gels was 60 to 200 @tg.°
The proteins were estimated
that were common to cytosol proteins were 17, 16, and
3 After
treatment
with
DNase
and
RNase,
the
recoveries
were
86.2,
86.3,
96.8, 86.2, and 87.7%, respectively, in the Novikaff hepatoma fractions a
through e (see “Abstract―).
For the corresponding fractions of normal liver,
the recoveries were 88.4, 91.7, 94.1 , 90.0, and 96.3%, respectively. Following
treatment with DNase and RNase, the protein patterns were essentially the
same as those without such treatment. Acid loading permits highly acidic
nucleic acids to migrate towards the acid side, whereas the proteins migrate
ontothegel.
4 After
dialysis,
the
recoveries
of
the
proteins
in
Fractions
a
through
e
(see
“Abstract―)
were, respectively, 72.5, 86.2, 94.3, 84.8, and 96.1% in the
normal liver and 84.2, 81.0, 92.9, 86.5, and 95.4% in the Novikoff hepatama.
The protein patterns obtained for the dialyzed, lyaphilized samples were very
similar to those obtained after ethanol precipitation.
5 To determine
the yields and distribution of the proteins in the gels, a
study was made on r°Slmethianine-labeled Novikoff hepatoma protein
508
fractions. The initial samples were dissolved in 20 @I
of isoelectric focusing
sample buffer in amounts ranging from 180 to 600 @g.
A 10-@laliquat was
placed on the gel, and 60 to 200 @sg
actually entered the isoelectric focusing
gels. Only 1.2% of actual loading samples was last in the acid buffer. The
distribution in the isoelectric focusing gels was: cap, 32 to 49%; main gel, 47
to 64%; and basic side, 0.5 to 2%, far mast fractions. Far the 3 M NaCI/7 M
urea fraction, the distribution differed; i.e. , the cap contained 32%, the main
gel contained 47%, and the basic side contained 21%, probably reflecting
the large amounts of histanes and other basic proteins in this fraction.
I The standard marker
proteins used were: actin, 46/5.7; a-amylase, 45.5/
5.6; Elongation Factor 1, 56/8.3; GAP-DH (subunit), 36/8.0; RNase T2, 35/5.
In our studies, the p1was 5.7 for chicken breast muscle actin (courtesy of Dr.
B. R. Brinkley) and rabbit skeletal muscle actin (U. S. Biachemicals,
Cleveland, Ohio). These comigrated precisely with the actin spat in our gel
patterns.
CANCERRESEARCHVOL. 39
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Two-Dimensional Electrophoretic
17%, respectively. This result indicates that there is very
limited, if any, cross-contamination of the nuclear and
cytosolfractions (otherthan byin vivo transport processes).
Patterns of Nuclear Proteins
among gels of 6 separate preparations. The analysis of
spots was performed on 6 sets of protein patterns obtained
from separate preparations.
Characteristicsof the Spots The spots shown in the
Numbersof SpotsIn Paftems. The proteinextractsfrom
figures are all proteins as indicated by their ausence follow
ing pronase treatment of the protein samples prior to 2dimensional gel electrophoresis. The spots were character
ized by p1, molecular weight, size, and density. To locate
the spots, a grid was prepared for each gel based on known
marker spots and marker spots in the samples. With this
grid, each spot was mapped in defined regions of molecular
weight and p1, and its size and density were evaluated from
0 to 4+ (Table 1A). The molecular weights in the SDS gels
were determined by measuring migration compared to
marker proteins of known molecular weight (Gallard
Schlesinger Chemical Manufacturing Corp., Canle Place, N.
Y.). The protein spots indicated in this paper are designated
by molecular weight (x 10@) and p1; i.e., Spot 44/5.7 had
an apparent molecular weight of 44,000 and a p1of 5.7.
the tumor contained 144 to 180 protein spots on the gels. A
total of 483 different spots were found on these gels; of
these, 252 spots were present in 2 or more fractions. Table
1A shows the number and percentage of major spots as
well as the major spots unique to each nuclear fraction of
the Novikoff hepatoma.
Table 1B shows corresponding results for normal liven.
These gels contained 108 to 200 distinct spots. A total of
427 different spots were found in the nuclear protein frac
tions. In both tissues, the smallest number of nuclear
protein spots was in the 3 M NaCI/7 M urea extract.
Major Spots in Patterns. In both tumor and liver, the
number of major protein spots was the largest in the 10 mM
Tnis extract (Table 1). In the tumor, the ratio of major
protein spots to total spots was highest in the 10 mM Tnis
fractions. The largest number of spots unique to a nuclear
fraction was found in the NaCI/EDTA extract in the tumor
and in the 0.6 M NaCI extract in the liver.
Some major proteins were present in one fraction only
(Table 1); the largest number of these were found in the 3 M
NaCI/7 M urea extract, i.e., 8 in the liver and 7 in the tumor.
In some instances, the major spots unique to a fraction
were the same for both tissues; for example, Spots 110/
8.25, 110/8.30, 106/8.40, and 59/8.50 were found in the 3 M
NaCI/7 M urea fractions of both Novikoff hepatoma and
normal liven.
RESULTS
Figs. 2A, 3A, 4A, 5A, 6A, and 7A show the 2-dimensional
gel patterns for proteins of the cytosol (Fig. 24) and the
sequential extracts of Novikoff hepatoma nuclear proteins
with 0.075 M NaCI/0.025 M EDTA (Fig. 34), 10 mM Tnis (Fig.
44), 0.35 M NaCI (Fig. 5A), 0.6 M NaCI (Fig. 6A), and 3 M
NaCl/7 M urea (Fig. 7A). The corresponding patterns for
extracts of normal liver are presented in Figs. 2B to 7B . The
reproducibility of the relative densities and positions of
spots for samples from individual preparations was excel
lent both for duplicate gels from a single preparation and
CommonCytoplasmicand Nuclear Spots. A numberof
spots were common to both cytoplasmic and nuclear frac
Table1
Protein spots in the cytosol and nudear liverUnique
fractions of the Novikoff hepatoma and normal
spotsFractionsTotal
of major
spotsNo. spotsc (%)@in
fractionsA.Novikoff
hepatomaCytosol15267(44.1)74
fraction
of major unique spots@'in nuclear
(%)bM.W./pI
(48.7)NaCI/EDTA1
7563
28/8.551
0 mM Tris1
.1)29/5.60.35
N NaCI1
(6.9)170/6.00.6
(36.0)51
8094
7468
(52.2)20
(39.1 )1
N NaCI15052
42/5.33
MNaCI/7M urea14451
77/6.4
(29.1 )97/5.6
31/7.4
31/8.0
55/5.2
87/5.7
48/5.1
32/7.0
(1 1
2
(36.7)37
(24.7)132/5.2
(35.1)35
(24.3)110/8.25
110/8.3
98/8.35
82/5.0
106/8.4
97/8.45
59/8.5
39/8.55B.Normal
liverCytosol10840
(52.8)NaCI/EDTA19173
(37.0)57
44/7.1lOmMTris17876(42.7)6
(38.2)38
(3.4)0.35
M NaCI20071
(9.5)0.6MNaCI17849(27.5)44(24.7)44/5.53
N NaCl/7 M urea12224
(19.9)78/7.4
a Tot@of
majorspots
51/7.2
45/8.2
110/8.3
106/8.4
59/8.5
(35.5)19
(19.7)42
(34.4)110/8.25
57/8.45
@
68/6.1
52/8.6
48/8.55
42/8.6
(%).
Total of spots
b Thta@
unique spo@
Total of spots
C
Only
the
spots
(2+
to
x 100(%).
4+)
were
included
in
this
group;
numbers
assigned
for
spot
density
and
size
were
4+
for
dense large spots, 3+ for densesmaller spots, 2+ for lessdense spots, 1+ for lessdensebut distinct spots.
FEBRUARY1979
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509
H. Takamiand H. Busch
tions (Table 2). In the Novikoff hepatoma, Spot 42/6.6 was
densest in the 10 mM Tnis fraction; Spots 59/6.0, 37/7.5,
and 37/7.7 were densest in the cytosol fraction; and Spot
63/6.3 was densest in the 0.35 M NaCI, 0.6 M NaCI, and 3 M
NaCI/7 M urea fractions. Two characteristic clusters of
proteins were found in the Novikoff hepatoma nuclei. One
was in the region of 45 to 63/5.0 to 6.3 in the 0.35 M NaCI,
0.6 M NaCI, and 3 M NaCI/7 M urea fractions, and the other
was in the region 37 to 41/7.5 to 8.0 in most nuclear
fractions; a similar cluster was found in the liven.
In the liver, Spot 78/5.8 was densest in the 0.6 M NaCI
extract and Spot 37/7.7 was densest in the NaCI/EDTA and
10 mM Tnis fractions, which differed from the result in the
tumor. Spot 63/6.3 was densest in the 0.35 M NaCI fraction
as in the tumor. Like Spot 37/7.7, Spots 47/8.25, 40/8.30,
and 37/7.5 were more dense in the NaCI/EDTA and 10 mM
Tnis fractions than in the other liver extracts.
NuclearProteinsCommonto SeveralFractions.Another
group of spots was found in all the nuclear fractions but
not in the cytosol (Table 2). In the tumor, the greatest
density and size of each of these spots was in the 10 mM
Tnis fraction, except for Spot 63/7.2. The 19 spots (Table 2)
were almost equally dense in several nuclear fractions.
Two spots, 82/6.8 and 82/7.1 , were found only in the 0.35
M NaCI,
0.6
M NaCI,
3 M NaCI/7
M urea
fractions
Table 3
Qualitatively different protein spots in Novikoff hepatoma and
normal liver
Thesedifferencesare fo spots 2 to 4+ with respect
density
in one tissue thatrwere abse+
tissue.Navikaff
At from the other
to size and
liverM.W./pI hepatamaNormal
FractionM.W./pl
Fraction170/6.0
0.35MNaCI
0.6MNaCI97/8.45
140/6.7
0.6MNaCI
140/6.8
0.6MNaCI
132/5.2 0.6MNaCI160/5.8
NaCI/urea
NaCl/EDTA
87/5.7
O.6MNaCI
84/6.3
Tris
82/5.0
0.6MNaCI98/5.0
0.35MNaCI62/6.2
94/7.8
94/7.9
0.6 M NaCI
61/6.1
NaCl/EDTA
0.6MNaCI
61/7.3
NaCI/EDTA78/7.4
NaCI49/5.3
76/8.4
NaCI/urea
72/5.5
65/5.2
NaCI/EDTA
0.6 M
NaCI/EDTA
0.6 M NaCI
48/5.1
0.6MNaCI
Tris38/7.3
48/7.9
Tris, 0.35 M
NaCI
42/5.3
0.35MNaCI
NaCI/EDTA,Tris
41/5.6
0.6MNaCI45/8.2
of the
Novikoff hepatoma. Four spots, 148/7.1 , 82/6.8, 82/7.1,
and 75/7.1 , were present only in the same 3 fractions of the
normal liven. These spots were densest in the 0.35 M
extract.
28/5.0
NaCITotal
27/4.9
NaCl/EDTA
Tris
28/8.45
Tris38/5.5
Tris
0.35 M
1812
KnownProteins.Recentstudiesinthislaboratory(21and
39; Rothblum L. I., Rao, M. R. S. and Busch, H., personal
communication) have shown that 3 protein spots, 56/8.3,
53/6.5, and 28/4.9, respectively, of the cytosol fraction of
the Novikoff hepatoma comigrated with subunits (EF-1a,
EF-1f3,and EF-1y) of EF-1H, the elongation factor of protein
synthesis (Figs. 2 to 4). EF-1a was also present in the NaCI/
EDTA
and
10 mM Tnis extracts.
Actin,
Spot
46/5.7,
was
Table 4
Quantitatively different protein spots in Novikoff hepatoma and
normal liver
These differences
are only for spots which were 4+ in one tissue
and 2+ or less in the other, or 3+ in one tissue and 1+ or less in
the other.
Novikoff hepatomaNormal
liverM.W./pIFractionM.W./pI
present as a dense spot in Novikoff hepatoma (@in Figs.
3A, 44, 5A, and 64) and as a less dense spot in most normal
97/5.6
NaCl/EDTA
liver patterns.
82/7.6 0.6MNaCI
82/7.7Fraction
0.6MNaCI76/8.20.35
Eight spots comigmating with the high-mobility-group
proteins (18) of calf thymus (generously provided by Dr. G.
M NaCI79/7.70.35
M NaCI,0.6 M
H. Goodwin and Dr. E. W. Johns of the Chester Beatty NaCI63/6.30.35
Institute) were found in small amounts in Regions 26.5/6—8
M NaCI,0.6 M
M NaCI, 0.6 M
NaCI79/7.8
NaCI
78/5.8
NaCI/EDTA,0.6 M
Table 2
NaCI
77/5.60.35
0.35MNaCI57/5.50.6
spotsCytosol
Commonprotein
and nuclear
fractionsNovikoffNovikoff
protein
fractionsNuclear
Normalhepatoma
liver108/5.7 liverhepatomaNormal
63/6.778/5.878/5.874/7.6148/6.9
57/7.767/7.1
70/7.771/8.396/8.1
55/6.363/6.363/6.368/7.296/8.2
51/5.759/6.047/8.2563/7.282/7.6
46/7.457/7.740/8.348/7.982/7.7
41/7.946/5.737/7.544/8.279/7.7
pratein
NaCI55/6.3NaCI/EDTA54/5.60.6
M
M NaCI,NaCl/
urea48/8.25NaCI/EDTA45/5.7Tris,
0.35 M NaCI,
0.6MNaCI,
NaCI40/7.7NaCl/EDTA44/8.2Tris37/7.7
NaCI/urea41/7.9NaCl/EDTA,
EDTA,0.35
M NaCI
37/7.9NaCI/
NaCI/EDTA,0.35
M NaCI, 0.6 M
40/7.542/6.641/7.976/8.2
37/7.742/6.879/7.8
40/7.737/7.537/7.975/6.9
37/7.937/7.730/7.964/8.1
510
0.6 M
NaCITotal613
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Two-Dimensional Electrophoretic
Patterns of Nuclear Proteins
nucleosome octamer (13, 19, 24, 25, 32, 35); the multiple
subunits of the RNA polymerases I, II, and Ill (8); and the 4
subunits of Elongation Factor EF-1H (Refs. 20 and 39;
Rothblum, L. I., Rao, M. A. S., and Busch, H., personal
communication). It is also likely that other types of proteins
Nuclear Proteins Unique to Normal Liver or Novikoff have subunit structures.
It is also important to note that some of the spots
Hepatoma. Eighteen proteins were found in one or more
nuclear extracts of Novikoff hepatoma that were not found probably represent variations in structural modifications of
in any fraction of normal liver (Table 3). These proteins proteins, such as amidation of glutamic and aspartic acid
have molecular weights ranging from 27,000 to 170,000. In residues and phosphorylation of senine and threonine resi
the fractions of normal liven, 12 spots were found in various dues.
Salt Extractions.The variationsin the proteinpatternsof
fractions that were not found in the Novikoff hepatoma
nuclear fractions (Table 3). The largest number of the the different nuclear fractions illustrate the efficient frac
different spots of the Novikoff hepatoma was in the 0.6 M tionation which is achieved by the salt extraction proce
dune. As noted earlier (24, 40), fractionation of the nucleo
NaCI extract. In the liven, the largest number of differences
lus by NaCI extractions effectively results in extraction of
was in the NaCI/EDTA extract.
QuantitativeDifferencesin ProteinsIn Liverand Tumor soluble RNA polymenase I in relatively dilute NaCI solutions,
NuclearFractions.Markedquantitativedifferencesin some extraction of the very lysine-nich histones in the 0.6 M NaCI
spots were noted in various fractions of the Novikoff hepa
extract (16, 17), and extraction of the nucleosomal core
toma and normal liven. As noted in Table 4, 6 spots ranging histones with 3 M NaCI/7 M urea (1, 4). These are just a few
in molecular weight from 44,000 to 76,000 were found in of the examples of differential extraction of the nuclear
much greaten density in various fractions of the Novikoff enzymes and structural proteins with NaCI solutions of
hepatoma than in the normal liven fractions and, conversely, different concentrations. Recently, it was found that the
13 different spots were found in much greaten density in the
normal liven fractions than in the Novikoff hepatoma.
tracted with 0.6 M NaCI (1). The numbers and types of the
restriction proteins in this important fraction are not yet
known.
DISCUSSION
Some proteins are specifically extracted with the different
Resolution of Nuclear Proteins. The steady and continu salt fractions. Such proteins could be portions of specific
structures on special synthetic enzymes. In agreement with
ing improvement in resolution with new analytical proce
dunes permits an increasingly refined analysis of nuclear the report of Comings and Okada (10), several proteins are
proteins of tumors and other tissues (3, 5). With the method distributed throughout most fractions. In the tumor and
liven, these proteins include Spots 37-41/7.5-8.0; these
of Omnicket a!. (37) using 2-dimensional gel electrophoresis,
it was possible
to identify a number of phosphoproteins
(15)
protein spots are dense and were found earlier by Liew et
a!.(23,28).
Level of Sensitivityof the Method. The levelsof spot
nuclear fractions of Novikoff hepatoma and other tissues
(43, 45). Although this method was considerably more visibility in the present study approach that needed for
visualization of proteins involved in gene control. O'Malley
efficacious than the 1-dimensional gel systems, particularly
for basic proteins such as proteins Al 1 and A24 and the and Means (36) have indicated that estrogen receptor pro
histones, its resolving power was less than satisfactory for teins are present in target cells in the order of several
proteins of higher molecular weight. The O'Famnellsystem thousand pen cell on cell nucleus. In this connection, the
(23, 28, 29, 33) and its minimodifications (20) have the yields of protein in the 0.6 M NaCI extract are appnoximately
advantage that a broader display of nuclear proteins is now 130 @g/gof tumor cells. In these gels (250 @g
protein), one
possible, although, underthe conditions used, the histones visualizes approximately 0.1 to 20 pg/spot derived from
migrate off these gels.
approximately 2 g of cells on 2 x 10°cells (approximately
The present “3-dimensional―
combination of extraction
130 fg/ceIl).
The spots containing
0.1
@gaccount
for 50 ag/
procedures and the 2-dimensional display (20, 33) show the cell. If one assumes that a molecule of protein (M.W.
largest number of stained nuclear proteins thus fan, i.e., 50,000) weighs 0.1 ag, there are approximately 500 to 600
483 proteins or polypeptides in the Novikoff hepatoma cell such molecules pen cell for a spot containing 0.1 @g.
nucleus and 427 proteins in normal rat liver nuclei (14). By Accordingly, the spots visualized in these patterns are
contrast, in the first review from this laboratory on the present in the 0.6 M NaCI fraction in abundances of approx
subject of nuclear proteins (5), only 3 main fractions had imately 500 to 100,000 molecules/cell. Thus, the possibility
been defined with any level of clarity: lysine-nich and angi exists that at least some of the differences found in the
nine-rich histones and “chromosin―
or “chromosomin.― proteins in the nuclear fractions reflect gene control fac
Multiple Spots from Individual Proteins. In view of the tons.
It is likely that more sensitive methods will be developed
earlier results from this and other laboratories (2, 44), it was
in the near future. Addition of tracers such as 32P,
not surprising that there are many species of polypeptides
in the cell nucleus, although the actual number of 480 is [3H]methionine, and 1251
will provide increased sensitivity of
greater than had been suggested in any of the earlier analysis of nuclear proteins. It is clear that an enormous
task confronts investigators, both in cell biology and in
reports (37, 43). Needless to say, several nuclear polypep
tides are protein subunits; e.g. , the 8 histones in the oncology, who will attempt to define the roles of these
and 26/7-9 of the NaCl/EDTA, 10 mM Tnis, 0.35 M NaCI, and
0.6 M NaCI fractions. Four such spots were present in the
Novikoff hepatoma pattern, and 5 such spots were present
in the normal liver pattern. Spot 26/8.15 was in the 10 mM
Tnis and 0.35 M NaCI patterns in both tissues.
‘
‘
restriction
.
and
stained
FEBRUARY
proteins
that
differed
in
chromatin
and
proteins―
of
nucleolan
rRNA
readouts
are
ex
other
1979
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1979 American Association for Cancer Research.
511
Two-Dimensional Electrophoretic Patterns of Nuclear Proteins
proteins. In addition, it is likely that the level of complexity
of the problem is greaten than that detected here. Studies in
progress on tumor nuclei show that 133 additional 32Plabeled spots that are not seen in stained patterns are
present (Wu, B. C. , Spohn, W. H. and Busch, H., personal
communication).
Tissue-specificNuclearProteins.Inasmuchasthe major
goal of our research is the determination of which of the
nuclear and nucleolar proteins are particularly involved in
cancer-related cell functions, it was of special interest to
evaluate the proteins that differ in normal and cancer cells.
Now that differences were found between the nuclear
proteins of the 0.6 M NaCI and 0.075 M NaCI/0.025 M EDTA
fractions of the rapidly growing Novikoff hepatoma and
normal liven, these fractions will be studied in regenerating
liven, fetal liver, and several Morris hepatomas of differing
growth rates. The goal of these studies will be to assess
which of the differences in proteins relate to rapid growth
and which, if any, are characteristic of neoplastic cells.
When common differences are found, such proteins will be
isolated from the Novikoff hepatoma which offers particular
advantages as a source for adequate amounts of these
nuclear macromolecules.
In the group of proteins most tightly bound to DNA and
extracted with 3 M NaCl/7 M urea, only Spot 97/8.45 was
present in the Novikoff hepatoma (Table 3) which was not
found in the normal liver. In the fraction that contains the
nucleolan “restriction
proteins,―the 0.6 M NaCI extract (1),
10 proteins were found in the Novikoff hepatoma that were
not found in the normal liver (Table 3). A total of 12 spots
were present in the liven that were different from those of
the tumor (Table 3). It is possible that such proteins are
gene control proteins.
The meaning of these differences requires further study;
e.g., do they relate to tumor growth or do they represent
isozymes that may have functions similar to those of the
normal liver? It is possible that at last the level of sensitivity
is sufficient to detect those proteins that interact with on
control the genes of tumor cells so that they continue to
grow and divide. These methods will be useful as analytical
techniques to follow purification of specific proteins and
polypeptides.
ACKNOWLEDGMENTS
The authors wish to express their great appreciation to Dr. Friedrich
Hirsch who developed many of the methods used in this study and to Katrina
N. NaIl who provided invaluable technical assistance in several phases of
this work. We are most appreciative to Rose K. Busch for supplying the
Novikoff hepatomas used in this study. The authors also thank Dr. B. C. Wu,
Dr. L. C. Yeoman, Dr. L. I. Rathblum, Dr. N. R. Ballal, and W. H. Spahn for
helpful suggestions.
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and
CANCER RESEARCH VOL. 39
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Two-Dimensional Gel Electrophoretic Comparison of Proteins of
Nuclear Fractions of Normal Liver and Novikoff Hepatoma
Hiroshi Takami and Harris Busch
Cancer Res 1979;39:507-518.
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