[CANCER RESEARCtt 35, 2954-2958, November 1975]
Fractionation of Nuclei and Analysis of Nuclear Proteins of Rat
Liver and Morris Hepatoma 7777 x
Brian W i l s o n , ~ M i c h a e l A. Lea, 3 Giorgio Vidali, and V i n c e n t G. AIIfrey 4
The Rockefeller University, New York, New York 10021
SUMMARY
The contributions of nuclear populations to the total
profile of nuclear proteins in a tissue were examined in
normal rat liver and Morris hepatoma 7777. Comparison by
sodium dodecyl sulfate polyacrylamide gel electrophoresis
of phenol-soluble nuclear proteins from tumor and control
liver revealed additional proteins of molecular weight
60,000, 100,000, and 135,000 and the loss of proteins of
about 45,000 and 55,000 in the tumor. Subfractionation of
liver nuclei on a 30 to 50% sucrose gradient yielded three
nuclear classes with nearly identical complements of the
phenol-soluble proteins. Similar fractionation performed on
the hepatoma nuclei also produced three nuclear populations. In the hepatoma nuclei, several differences in the
phenol-soluble proteins were found between the minor,
slowly sedimenting nuclear fraction, and the two major
fractions, while the two latter fractions were very similar in
their protein composition. Histones derived from both
tissues were also compared electrophoretically, indicating a
decrease in the concentration of histone H i " in all nuclear
classes derived from the tumor.
nonhistone nuclear proteins in liver and Morris hepatomas
were first reported by Chae et al. (8). These observations
were extended in other reports (2, 9, 14, 22, 24, 26) which
agreed on the presence of changes in hepatomas but differed
on their extent. Less notable changes have been recorded for
histones in liver neoplasms but alterations in side-chain
modification, rates of synthesis, and the amount of H I "
histone have been reported (3, 7, 15).
In view of the greater cellular heterogeneity in normal
liver than in hepatomas, it was possible that reported
differences in nuclear proteins reflected altered nuclear
populations rather than changes in a given type of nucleus.
Gonzalez-Mujica and Mathias (12) have shown by means of
sucrose gradient centrifugation that normal liver nuclei can
be separated according to ploidy and cell type, and these
workers have reported that the individual nuclear types have
nonhistone chromosomal proteins giving different electrophoretic patterns. We have undertaken studies of this type
with normal liver and Morris hepatoma 7777 in order to
obtain information on the nuclear types in hepatoma which
may be responsible for overall changes in nuclear proteins.
M A T E R I A L S AND M E T H O D S
INTRODUCTION
The postulated role of chromosomal proteins as regulators of genetic expression has prompted many investigations
of the tissue specificity of these proteins (1). The growth of
neoplastic cells might conceivably be influenced by changes
in the relative amounts of different nuclear proteins, and
this possibility has led several investigators to examine
chromosomal proteins in cancer cells. The Morris series of
hepatomas show a spectrum of growth rates that provide a
useful model for the correlation of biochemical changes with
tissue growth. Differences in the electrophoretic patterns of
'This work was supported by USPHS Grants CA-12933, CA-14908,
and CA-16274 and by American Cancer Society Grant VC-114D.
2On sabbatical leave from the MRC Iodine Metabolism Research Unit,
Department of Pharmacology, University of Stellenbosch Medical School,
Ti'ervlei, South Africa.
Present address: Department of Biochemistry, College of Medicine
and Dentistry of New Jersey, New Jersey Medical School, Newark, N. J.
07103.
' To whom reprint requests should be addressed.
Received April 29, 1975; accepted July 18, 1975.
2954
Animals and Tumors. Experiments were performed with
male Buffalo rats. Morris hepatoma 7777 was maintained
as s.c. transplants. The induction, histology, and growth
properties of this tumor have been described (19).
Isolation of Nuclei. Rats weighing 160 to 200 g were killed
by decapitation. Livers and tumors were immediately
removed and chilled. All adhering connective tissue and
grossly necrotic regions of the tumor were removed before
the tissues were finely minced with scissors. Twenty g of
minced tissue were homogenized in 100 ml of 50 mM
Tris-HCl, pH 7.7, containing 0.32 M sucrose and 25 mM
MgCI2, using sequential shearing in a Teflon-to-glass homogenizer with pestle clearances of 0.20 and 0.30 ram. Six
to 8 strokes were applied with the loose pestle, and 10 to 12
strokes were applied with the tight pestle at a rotation speed
of 1200 rpm. The homogenates were filtered through 8
layers of cheese cloth and centrifuged at 750 • g for 6 min.
The crude nuclear pellet was resuspended in 15 volumes 2.4
M sucrose containing 3 mM MgCI2 and was centrifuged at
130,000 x g for 75 min. The 2.4 M sucrose solution and all
subsequent sucrose solutions used were adjusted to pH 7.4
with i M NaHCO3. The purified nuclei were washed in 10
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Nuclear Proteins of Hepatoma
volumes of buffered 15% sucrose containing 1 mM MgCI2
and were centrifuged at 500 • g for 7 min. The nuclear
pellet obtained was gently suspended in a final volume of 5
ml of 15% sucrose with a Vortex mixer.
Fractionation of Nuclei. A modification of the method of
Johnston et al. (13) was used for the fractionation of nuclei.
One-ml aliquots of the nuclear suspension were layered on
30-ml linear sucrose gradients (30 to 50%) in 1- x 3-inch
cellulose nitrate tubes. The gradients were immediately
centrifuged at 1250 rpm (200 • g) for 10 min at 5 ~ in a
Sorvall centrifuge (Model RC2-B, equipped with zonal
rotor controls) with a HS-4 rotor. Both autobrake and
compressor were switched off during the centrifugation.
Once the purified nuclei were suspended in the 15% sucrose,
it was found necessary to perform the gradient centrifugation as quickly as possible to prevent aggregation of the
nuclei. The gradients were fractionated into l-ml fractions,
and the absorbance at 280 nm was read. In some cases,
gradients were monitored at 600 nm or by the diphenylamine assay for DNA (6). The profiles obtained were found
to be essentially the same as those read at 280 nm. The
fractions constituting the individual zones were pooled as
indicated in Chart I.
Extraction of Proteins from Nuclear Fractions. The
different classes of nuclei separated by sucrose gradients
were washed 3 times with 0.25 M HCi. The 1st 2 washes were
combined, and the extracted histones were precipitated by
adding 10 volumes of acetone. The nuclei were then washed
once with 1:1 (v/v) chloroform:methanol containing 0.2 M
HC! and once with 2:i (v/v) chloroform:methanol containing 0.2 M HCI. Phenol-soluble proteins were extracted from
the washed nuclei according to the procedure of Teng et al.
(25). The extracted proteins were restored to the aqueous
phase as described by Shelton and Allfrey (23).
Electrophoresis of Nuclear Proteins. Histones were characterized by polyacrylamide gel electrophoresis in 0.9 y
acetic acid and 6.25 M urea by the method of Panyim and
Chaikley (20). Fifty ,ug protein, measured by the method
of Lowry et al. (18), were applied to each gel. The protein
bands were stained with 0.1% Amido black in methanol:
acetic acid:water (3:1:6). The gels were destained in the
same solvent and scanned in a densitometer, measuring absorbance at 615 rim. Nonhistone proteins were analyzed by
electrophoresis in 8.8% polyacrylamide gels containing 0.1%
SDS 5 as described by LeStourgeon and Rusch (17).
Prior to the electrophoresis, the nonhistone proteins were
dialyzed for 24 hr against 2 changes of 0.01 M sodium
phosphate buffer, pH 7.4, containing 0.1% SDS and 0.14 M
2-mercaptoethanol. Approximately 90 ~ag protein, determined by the method of Christian and Warburg (10), were
applied per gel, and electrophoresis was carried out at 2.25
ma/gel for about 6 hr. The gels were stained with 0.1%
Coomassie brilliant blue R in isopropyl alcohol:acetic
acid:water (2.5:i:6.5). The protein bands were analyzed by
densitometry at 590 nm. Estimates of the molecular weights
of the individual nonhistone protein bands were based on
s T h e a b b r e v i a t i o n used is: S D S , sodium dodecyl sulfate.
I
2
3
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(a)
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1.0
0.5
/,
E
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Z
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rY
0
oo
rn
(b)
I
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I.,5
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0.5
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i
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30
FRACTION NOI
C h a r t 1. A b s o r b a n c e profiles of nuclei s e p a r a t e d by c e n t r i f u g a t i o n on
30 to 50% sucrose gradients. T h e nuclei were obtained f r o m (a) control rat
liver or (b) h e p a t o m a 7777. T h e fractions that were pooled to give the 3
nuclear classes are indicated by a r r o w s .
mobility versus molecular weight plots for proteins of
known molecular weight under identical conditions.
RESULTS
Isolation and Fractionation of Nuclei. Sucrose gradient
profiles showed that normal liver nuclei isolated from
Buffalo rats weighing 160 to 200 g separated into 3 distinct
classes on 30 to 50% sucrose gradients (Chart la). Similar
findings were previously reported for Norwegian hooded
rats by Johnston et al. (13) who described Zone 1 as stromal
diploid nuclei, Zone 2 as parenchymal diploid nuclei, and
Zone 3 as parenchymal tetraploid nuclei. Nuclei isolated
from the rapid growing and poorly differentiated Morris
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2955
B . W i l s o n et al.
h e p a t o m a 7777 were s i m i l a r l y found to separate into 3
distinct zones (Chart 1B). The inclusion of 2 mM CaCI2 in
the h o m o g e n i z i n g m e d i u m was observed to increase the
yield of t u m o r nuclei. A l t h o u g h Z o n e 1 of the h e p a t o m a
cosedimented with Zone 1 of the liver nuclei, Zones 2 and 3
of the h e p a t o m a nuclei sedimented a little faster than the
corresponding zones of liver nuclei. The proportion of nuclei
found in Z o n e 1 was very m u c h less in the h e p a t o m a than in
n o r m a l liver. On the other hand, a greater proportion of
nuclei was observed in Z o n e 3 in the case of the tumor.
N u c l e a r profiles obtained from host liver were the same as
those from the livers of n o n - t u m o r - b e a r i n g rats.
Acid-soluble Proteins. C h a r t 2 records the densitometric
tracings for u r e a - p o l y a c r y l a m i d e gel electrophoresis of
acid-soluble proteins extracted from the 3 different classes
of liver and h e p a t o m a 7777 nuclei separated on 30 to 50%
sucrose gradients. In all cases, the m a j o r histone components exhibited similar patterns with respect to a m o u n t and
relative migration. The m i n o r histone fraction H1 ~ was
present at a similar concentration in all the nuclear fractions
obtained from n o r m a l liver, but was greatly reduced or not
detectable in the 3 nuclear classes from the tumor.
Phenol-soluble Proteins. The phenol-soluble proteins extracted from the residue r e m a i n i n g after acid extraction of
the nuclei were analyzed by electrophoresis according to
m o l e c u l a r weight on S D S - p o l y a c r y l a m i d e gels. Chart 3
shows the gel patterns of the proteins extracted from
A
5
1:55,000
I00,000
80,000
65,000
55,000 -~
45,000
Chart 3. SDS-polyacrylamide gel electrophoretic patterns of phenolsoluble proteins extracted from unfractionated nuclei of livers from
non-tumor-bearing rats (I), host livers (2), and Morris hepatoma 7777 (3).
Values for molecular weights are indicated to the left of the gels.
D
B
E
o
~z
J
JL
F
JL
Chart 2. Polyacrylamide gel electrophoretic patterns of histones obtained from hepatoma 7777 (A, B, and C) and control rat liver (D, E, and
F). The histories were extracted from nuclei of Class 1 (A and D), Class 2
(B and E), and Class 3 (C and F). Where present in significant amounts, the
position of histone H l ~ is indicated by an arrow.
2"956
2
unfractionated nuclei of control and host liver and also of
M o r r i s h e p a t o m a 7777. N o m a j o r differences were observed
in the overall protein patterns. However, in the tumor, there
were both additions and deletions of single bands. In the
high-molecular-weight region, several additional protein
bands were evident. At 135,000 daltons and also at about
100,000 daltons, additional bands were observed. In the
lower-molecular-weight range, additional bands were found
at a p p r o x i m a t e l y 65,000 daltons. F u r t h e r m o r e , there was
the deletion of a protein band at 55,000 dattons; also, at
45,000 daltons, a band appeared to be greatly reduced or
absent. These alterations were highly reproducible from one
preparation to another. There were no significant differences between the phenol-soluble proteins from the livers of
n o n - t u m o r - b e a r i n g and host rats.
Rat liver nuclei were separated into the different classes
on a 30 to 50% sucrose gradient, and the phenol-soluble
proteins were extracted and analyzed on S D S polyacrylamide gels, as already described. In C h a r t 4, the corresponding
protein patterns of the individual nuclear classes are compared with the proteins extracted from unfractionated liver
nuclei. It appears that the electrophoretic patterns of the
proteins from the 3 nuclear classes isolated from liver are
very similar to the protein pattern of unfractionated nuclei.
Similarly, the phenol-soluble proteins extracted from the 3
classes of t u m o r nuclei were c o m p a r e d with unfractionated
t u m o r nuclei by m e a n s of S D S - p o l y a c r y l a m i d e gel electrophoresis (Chart 5). Except for a few m i n o r quantitative
differences, the phenol-soluble protein c o m p l e m e n t of nuclear Classes 2 and 3 of the t u m o r seemed to be similar to
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Nuclear Proteins 0[" H e p a t o m a
I
Y
I
2
3
,3s,ooo-IO0,O00--80,000
--
65,000
--
55,000
45,000
Chart 4. SDS-polyacrylamide gel electrophoretic patterns of phenolsoluble proteins extracted from unfractionated liver nuclei (T) and from the
3 classes of liver nuclei fractionated on 30 50% sucrose gradients: 1, Class
1 nuclei; 2, Class 2 nuclei, and 3, Class 3 nuclei. (See Chart 1 for
explanation of nuclear classes.)
Chart 5. SDS-polyacrylamide gel electrophoretic patterns of phenolsoluble proteins extracted from unfractionated nuclei of Morris hepatoma
7777 (T) and from the 3 classes of hepatoma nuclei fractionated on 30 to
50% sucrose gradients: I, Class 1 nuclei, 2, Class 2 nuclei, and 3, Class 3
nuclei. (See Chart 1 for explanation of nuclear classes.)
that extracted from unfractionated tumor nuclei. Although
the additional proteins observed in the tumor in the
molecular weight regions of 135,000, 100,000, and 65,000
daltons are present to a lesser extent in nuclear Classes 2
and 3, they appear to be enriched in Class 1 nuclei of the
tumor. Interestingly, these proteins are the ones lacking in
the liver nuclei. Furthermore, proteins of molecular weights
of approximately 80,000 and 45,000 that are present in
nuclear Classes 2 and 3 are greatly reduced in Class 1 nuclei.
These observations contrast with the uniformity of protein
patterns derived from the 3 nuclear classes from normal
liver.
comparison of our results with those of other workers is
complicated by the different protein extraction techniques.
In studying the biochemistry of liver carcinogenesis, it is
important to take into account the complex cellular structure of this organ. While in normal adult rat liver the nuclei
from stromal cells are diploid, most of the parenchymal
cells may be either diploid or tetraploid, according to the
age of the animal (13). It is appropriate, therefore, to assess
the influence of different nuclear classes on normal and
abnormal biochemical activities of the liver. GonzalezMujica and Mathias (12) have reported that the electrophoretic patterns of nonhistone chromosomal proteins vary
within the different classes of liver nuclei fractionated on 20
to 50% sucrose gradients. Furthermore, these workers
showed that thioacetamide-induced alterations in nonhistone proteins of liver varied according to nuclear class.
Baserga (5) has suggested that the heterogeneity of cell
population makes a comparison between liver and hepatomas somewhat meaningless. Therefore, it would be important to establish whether the differences previously reported
in the electrophoretic patterns of nonhistone proteins from
various Morris hepatomas are due to heterogeneity of
nuclear type and whether specific alterations are limited to
any particular class of nuclei. Accordingly, in our study of
the chromosomal proteins from the fast-growing, poorly
differentiated Morris hepatoma 7777, we developed a
procedure for the fractionation of the hepatoma nuclei into
3 different classes as previously established for liver nuclei
(13). Analysis by SDS-polyacrylamide gel electrophoresis
of the phenol-soluble proteins extracted from the 3 individual classes of liver nuclei showed similar banding patterns.
DISCUSSION
The comparison of SDS electrophoretograms of proteins
obtained from total nuclear populations of liver and Morris
hepatoma 7777 shows that there are several additional
bands appearing in the tumor and also a deletion of at least
1 protein band. These changes cannot be attributed to
contamination by membrane or cytoplasmic debris since the
differences still exist when nuclei are washed with 1% Triton
X-100 (data not shown). It is also unlikely that all these
changes are due to differences in contractile proteins, as
reported by LeStourgeon et al. (16) for nondividing versus
proliferating slime molds. The additional bands in the
hepatoma nuclear protein pattern are not in the molecularweight regions of contractile proteins.
Changes in nonhistone nuclear proteins in liver neoplasia
have also been reported by others (2, 9, 14, 22, 24, 26), but
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2957
B. Wilson
e t al.
W i t h i n 1 t i s s u e , t h e o n l y n o t a b l e c h a n g e s e e n in a n u c l e a r
f r a c t i o n w a s in t h e m i n o r n u c l e a r f r a c t i o n o f t h e t u m o r
( Z o n e 1) w h i c h w o u l d c o n t r i b u t e little t o t h e t o t a l p r o f i l e f o r
the tissue. However, our experiments do not exclude the
possibility that tumor nuclei of Zone 1 are derived from
n o n t u m o r cells. It m a y be n o t e d t h a t t h e s e n u c l e i c o s e d i m e n t w i t h t h e s t r o m a l d i p l o i d n u c l e i o f c o n t r o l liver. T h e
acid-soluble proteins also showed intratissue consistency,
b u t t h e r e w a s a n i n t e r t i s s u e d i f f e r e n c e in t h e H 1 ~ h i s t o n e .
T h i s h i s t o n e , w h i c h is f r e q u e n t l y d e c r e a s e d in r a p i d l y
d i v i d i n g cells (21), h a s a l s o b e e n d e s c r i b e d as h i s t o n e fla
(15), F r a c t i o n A l l (4), o r A (1 1). In v i e w o f t h e e s s e n t i a l
s i m i l a r i t y in n u c l e a r p r o t e i n s o b t a i n e d f r o m d i f f e r e n t liver
n u c l e a r p o p u l a t i o n s at t h e level o f r e s o l u t i o n o b t a i n e d u n d e r
o u r c o n d i t i o n s , it a p p e a r s t h a t c o m p a r i s o n s c a n be m a d e
between liver and hepatomas, which are not made meaningless b y t i s s u e h e t e r o g e n e i t y .
ACKNOWLEDGMENTS
We are indebted to Dr. Harold P. Morris for providing tumor-bearing
rats from which hepatoma 7777 was transplanted in these studies.
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CANCER
RESEARCH
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VOL. 35
Fractionation of Nuclei and Analysis of Nuclear Proteins of
Rat Liver and Morris Hepatoma 7777
Brian Wilson, Michael A. Lea, Giorgio Vidali, et al.
Cancer Res 1975;35:2954-2958.
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