[CANCER RESEARCH 33, 1169-1176,
June 1973]
A Comparative Study of Some Properties of Chromatin from Two
"Minimal Deviation" Hepa tomas1
Eugene A. Arnold,2 Mary Margaret Buksas, and Keith E. Young
Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
SUMMARY
Chromatin has been isolated from slow-growing Morris
hepatoma 96ISA, which has a normal chromosome number
and normal karyotype. Some of the compositional and
functional properties of this chromatin have been examined
and compared with those of the chromatin from normal liver
and from fast-growing Morris hepatoma 5123C. The chromatin
of both hepatomas was found to have a slightly greater
content of nonhistone protein and RNA, an increased rate of
incorporation of amino acid into histones, and a reduced
turnover of nonhistone protein, compared to the chromatin
from normal liver. The chromatin from hepatoma 9618A had
the same in vitro template activity for RNA synthesis as did
chromatin from liver, and the electrophoretic pattern of the
nonhistone proteins was qualitatively identical to the pattern
obtained with liver nonhistone proteins. The chromatin from
hepatoma 5123C, on the other hand, exhibited a marked
increase in template activity, compared to that from normal
liver, and there were several alterations in the electrophoretic
pattern of the nonhistone proteins. The data suggest that some
of the differences between the chromatin of rapidly growing
hepatomas and that of normal liver are a feature of malignant
progression rather than the process of neoplastic transforma
tion.
INTRODUCTION
Several comparative studies have been made of the
compositional and in vitro functional properties of chromatin
from neoplasms versus the properties of chromatin from
normal tissues. The purpose of these studies was to find
differences that might provide an understanding of the
neoplastic state. However, many of these studies were done
with poorly differentiated tumors or tumors with primary cells
of origin that are in doubt (such as the Novikoff ascites
tumor, Walker 256 carcinosarcoma, or Jensen sarcoma). With
such anaplastic tumors, differences in compositional and
functional properties may arise only as features of progressive
cancer and may not be fundamental changes essential to
neoplastic transformation (34). For this reason we studied
isolated chromatin from the Morris hepatoma 9618A, the
1This investigation was supported by Grant CA08306 from the
USPHS, National Cancer Institute, Bethesda, Md.
1Recipient of Research Career Development Award 1-K04CA38871-01A1 from the National Cancer Institute, Bethesda, Md.
Received December 29, 1972;accepted February 26, 1973.
morphology of which is similar to that of normal hepatocytes
and which is reported to have a normal chromosome number
and normal karyotype (29). This hepatoma has been described
as "karyotypically
one of the two least deviated minimaldeviation hepatomas available" (35). For purposes of compari
son, we also examined chromatin from the relatively rapidly
growing Morris hepatoma 5123C which has been studied
previously (15,30,37).
We determined the total histone, total nonhistone, and total
RNA content of the chromatins. We measured the relative rate
of incorporation of labeled amino acid into the histone and
nonhistone proteins at 2 different time periods to obtain an
estimate of the relative turnover of the 2 fractions. The
capacity of the chromatins to serve as templates for the
synthesis of RNA in vitro was assayed and, as a major goal of
the study, we isolated and characterized electrophoretically
the nonhistone or acidic proteins of the chromatins. The
nonhistone proteins have recently been implicated in the fine
control of gene expression (14, 39, 43) and reportedly show
changes in tumors (21, 22, 41) and show tissue specificity in
general (21, 25, 27, 33, 44). The classes and quantities of
histones, on the other hand, are very similar in both normal
and malignant cells (2, 15, 18, 23, 40) and may even have
identical amino acid sequences (24,45).
Aiding in these studies were our improved methods
(developed in this laboratory) for isolating and purifying
chromatin by a gentle, nonshearing process and for isolating
chromosomal proteins without using extremes of pH or
protein-denaturing agents (1).
MATERIALS AND METHODS
Animals and Tumors. Morris hepatomas 9618A and 5123C
were maintained by i.m. transplantation into male Buffalo
rats. Animals bearing hepatoma 96ISA were killed 60 to 70
weeks after inoculation, and those bearing hepatoma 5123C
were killed 7 to 9 weeks after inoculation. Generations 7 and 8
of hepatoma 9618A and Generations 87 through 93 of
hepatoma 5123C were used in these experiments.
Preparation of Chromatin. Animals were killed by decapita
tion, and livers and tumors were removed and placed in
ice-cold 0.25 M sucrose-0.05 M Tris-HCl (pH 7.5):0.025 M
KC1: 0.005 M MgCl2. All subsequent operations were
performed at 0—4°.
After adhering connective tissue and gross
necrotic regions were removed, the tissues were homogenized
and nuclei were isolated by pelleting through 2.2 M sucrose at
pH 5.9, by the method of Grunicke et al. (15), except that the
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1169
E. A. Arnold, M. M. Buksas, and K. E. Young
CaCl2 (0.004 M) was replaced by an equimolar amount of
MgCli which produced a cleaner nuclear pellet with less
clumping. In some experiments, the purified nuclei were given
an additional wash in 1% Triton X-100, as described by Blobel
and Potter (3). Purified chromatin was prepared as previously
described (1), be extraction of the nuclei with 0.075 M NaCl,
EDTA, and by subsequent washing of the crude chromatin
with Tris buffers of decreasing ionic strength. For determina
tion of chemical composition and template activity, the
chormatin was solubilized in distilled water adjusted to pH 8.0
with ammonium hydroxide. For isolation of chromosomal
proteins, the final suspension of washed chromatin was
adjusted to 0.01 M Tris-HCl, pH 8.0, and the condensed
chromatin was recovered by centrifugation at 10,000 X g for
20 min.
Isolation of Chromosomal Proteins. Total chromosomal
proteins were isolated by dissociation of the chromatin in 2.5
M guanidinium chloride, and removal of DNA was as
previously described (1), except that the dissociation step was
done in the presence of 0.05 M NaHSO3 and 0.01 M
ß-mercaptoethanol (32). Separation of the protein preparation
into histone and nonhistone proteins was accomplished on a
column of SE-Sephadex by a modification of the method
described previously (1). A stepwise salt-elution gradient was
used for convenience and 0.01 M (3-mercaptoethanol was
incorporated into the buffer system. For details of the elution
procedure, see Chart 2.
Electrophoresis of Chromosomal Proteins. Total chromo
somal proteins were analyzed on 15% acrylamide gels at pH
2.7, by the method of Panyim and Chalkley (32). Samples
were prepared for electrophoresis as described previously (1).
Nonhistone proteins were analyzed also by the method of
Panyim and Chalkley, but on gels in which the acrylamide
concentration was reduced to 7.5%, the bisacrylamide con
centration raised to 0.25%, and the electrophoresis run for 3
hr. Nonhistone protein samples were prepared in the same
manner as were the total chromosomal proteins.
Template Activity of Chromatin. In vitro RNA synthesis on
chromatin or DNA templates was measured in the assay
medium of Burgess (6). Each reaction mix (0.25 ml) contained
9 units of Escherichia coli RNA polymerase (obtained from
Miles Laboratories, Kankakee, 111.).Before the assay, both liver
and hepatoma chromatins were sheared by 5 passages of the
chromatin through a 20-gauge needle. This was found to be
the minimal number of passages required for complete
solubilization of hepatoma chromatin in the assay medium.
Determination of Specific Activities of Protein Fractions.
Rats received i.p. injections of 0.5 mCi L-leucine-4,5-3H (51
Ci/mmole; Schwarz/Mann, Orangeburg, N. Y.) either 1 or 24
hr before sacrifice. Leucine was chosen for these studies
because total histones and nonhistone proteins of liver contain
about equal proportions of this amino acid. Radioactivity of
protein in total-cell homogenates or suspended nuclei was
determined by the filter-paper-disk method of Bollum (4).
Radioactivity of histone and nonhistone protein fractions was
determined directly by incorporation of an aliquot of the
protein solution (in 5 M urea) in a toluene cocktail containing
Beckman BBS-3 solubilizer. Radioactivity of both the disks
and liquid samples was counted in a Beckman LS-250 liquid
1170
scintillation counter to a S.D. of ±2%.Protein content of the
samples was determined by the method of Lowry et al. (26),
with serum albumin or calf thymus histone as standard.
Chemical Composition of Chromatin. DNA, RNA, total
protein, histone protein, and nonhistone protein of chromatin
were determined as described previously (1). Absorption
spectra were measured on a Gilford Model 2000 spectrophotometer.
RESULTS
General Observations and Chemical Composition. Yields of
chromatin from small and young 9618A hepatomas were
approximately the same as those from normal liver (about
18 A26o units per g tissue) but dropped as low as 50% in
older and larger tumors, compared with those from liver.
Yields from 5123C hepatomas varied from 20 to 50% of those
from normal liver. Chromatins from both 9618A and 5123C
hepatomas were less soluble in distilled water than liver
chromatin. Both tumor chromatins required shearing to
prevent precipitation in the assay medium used to measure
template activity, while liver chromatin was soluble without
shearing. This tendency of tumor chromatin to be more
insoluble has been noted before in the case of the 5123C
hepatoma (37). UV absorption spectra of the tumor and liver
chromatins are nearly identical and correspond to published
spectra for normal rat liver chromatin (28). The only
difference noted between the hepatoma and liver spectra was a
small decrease (about 5%) in the 260:240 nm ratio, which
probably reflects the increased protein to DNA ratio in these
tumor chromatins as discussed below. The chemical composi
tion of the chromatins is given in Table 1. There is
approximately a 50% increase in the amount of nonhistone
protein and RNA in both the 9618A and 5123C chromatins,
compared with liver chromatin. The amounts of total protein
and RNA present in the 5123C chromatin are in agreement
with those reported by Smith and Mora (37). Treatment of the
nuclei with Triton X-100 did not affect the nonhistone:DNA
ratios. For all chromatins, the amount of histone remains
essentially constant at a 1:1 ratio with DNA, which confirms
the finding of others that the amount of histones in
minimal-deviation hepatomas does not differ significantly
from that in normal tissues (15, 37, 40).
Capacity to Serve as a Template for in Vitro RNA
Synthesis. The template activity of the liver and hepatoma
chromatins is shown in Chart 1. The activities of liver and
9618A hepatoma chromatin at the saturation level do not
differ significantly. The activity of the 5123C chromatin
shows, on the other hand, a marked increase over that of liver
chromatin. All 3 chromatins exhibited insignificant activity
without added polymerase. At saturation values of DNA input,
the activity of liver chromatin was approximately 6% of that
of purified DNA. The template concentration required for
half-maximal reaction velocity was calculated with the use of a
double-reciprocal plot of 1/RNA against 1/DNA. The values
obtained were: normal liver, 15.4 jug DNA; hepatoma 9618A,
20.8 jug DNA; and hepatoma 5123C, 30.3 /ag DNA. All values
are expressed per 0.25 ml of incubation mixture. Since the
CANCER RESEARCH VOL. 33
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Study ofChromatin from Hepatomas
Table 1
Chemical composition of isolated chromatin from rat liver and Morris hepatomas 9618A and 5123C
DNA, RNA, total protein, and histone protein were determined as described in text. Nonhistone
protein was calculated by subtracting histone protein from total protein. (Some of the nonhistone
protein values were confirmed by actual analysis.) Each analysis was made on a chromatin preparation
derived from the pooled tissue of at least 3 rats.
Protein:DNA
Tissue
RNA:DNA
Histone:DNA
Nonhistone: DNA
liverNormal
Normal
washa)Host
liver (Triton
ISA)Host
liver (96
123C)Host
liver (5
liver (5123C) (Triton wash0)1.681.621.711.641.670.030.040.040.060.04.1.0.1.1.10.60.60.60.50.6
Hepatoma9618A
2.06±0.03b
0.07 ±0.02
1.2+0.04
0.9 ±0.07
Hepatoma5123C
Hepatoma 5123C (Triton wash0)
2.00 + 0.21
2.39
0.12 ±0.04
0.03
1.1 ±0.20
1.0
0.9 ±0.05
1.4
a Nuclei from which chromatin was prepared were washed in 1% Triton X-100, as described in text.
0 Mean ±S.E.
50
o
5123C
§ 400
o
HI
t- 300
oc
O
« 200
O
u
100
O
10
DNA
20
INPUT
30
40
lUGI
Chart I. Template activity for RNA synthesis of chromatin from
normal liver and hepatomas 96ISA and 5123C. Activities were
measured at increasing concentrations of chromatin template until
saturation conditions were reached. Activity is expressed as pmoles of
ATP-'4 C incorporated into trichloroacetic-acid-insoluble
material
following 10 min of incubation at 37°
saturation curves indicate the amount of template required to
titrate the available polymerase (5), these values indicate that
the frequency and availability of binding sites for the RNA
polymerase are increased 2-fold in the 5123C chromatin but
are increased only slightly in the 96ISA chromatin.
Isolation and Comparative Electrophoretic Characterization
of Nonhistone Proteins. Fig. 1 shows the electrophoretic
pattern of the total chromosomal proteins isolated from
normal liver and the 2 hepatomas. The major bands indicated
by arrows are the 5 major classes of histone (32), and there
appears to be little qualitative difference between the patterns
of the 3 tissues. There is a slight increase in a fast-moving
broad band in the tumor patterns, but histone degradation
products are known to migrate as fast-moving bands in this
area. Small quantitative differences would not be detected on
JUNE
Fig. 1. Electrophoretic patterns of total chromosomal proteins from
normal liver and hepatomas on 15% acrylamide gels, pH 2.7. Bands
indicated by arrows are histones. Lighter bands in upper one-half of gel
are nonhistone proteins. Gel on left shows proteins from normal liver,
middle gel shows proteins from hepatoma 96ISA, and gel on right
shows proteins from hepatoma 5123C. About 150 ng of protein were
applied per gel.
1973
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1171
E. A. Arnold, M. M. Buksas, and K. E. Young
the gels. The nonhistone proteins appear in the upper one-half
of the gel, and although the patterns appear basically similar,
they are too complex a mixture for small differences (if
present) to be seen. For this reason, the nonhistone proteins
were separated from the histones on a preparative scale and
were reexamined in a gel system designed to give optimal
resolution. Chart 2 shows the separation of the nonhistone
proteins from the histones on a column of SE-Sephadex. The
elution patterns of the proteins from liver and hepatoma
96ISA were similar. However, when the 5123C chromosomal
proteins were fractionated, a small protein peak eluted just
before the main nonhistone protein peak. The yield of protein
from this minor peak was very small, and although it has been
found to contain only a few nonhistone proteins, it has not
yet been adequately characterized. The pooled and con
centrated main nonhistone protein fraction was analyzed on
7.5% acrylamide gels. This electrophoresis system is ideally
suited for the separation of nonhistone proteins, because any
contaminating histone present will migrate out of the gels.
The electrophoretic patterns are shown in Fig. 2. The
nonhistone proteins from hepatoma 9618A and liver have
nearly identical patterns. Within the limits of a slight
variability from one preparation to another in some faint
bands from both tissues, no reproducible qualitative differ
ences could be detected in the electrophoretic patterns, which
clearly show over 20 distinct bands. However, the pattern of
nonhistone proteins from hepatoma 5123C showed major
changes, with both additions and deletions of bands, compared
to the pattern of liver nonhistone proteins. There was some
variability of the patterns of the 5123C proteins from one
preparation to another. Some of this variability may result
from degradation of protein occurring during the isolation
procedure. When NaHS03 or 0-mercaptoethanol was omitted
during the isolation steps, the 5123C nonhistone proteins
often were severely degraded, as evidenced by fewer and more
diffuse bands on electrophoresis. On the other hand, the
96ISA and liver nonhistone proteins gave the same patterns
with or without the use of the protein protecting reagents.
Amino Acid Incorporation
into Chromosomal Protein
Fractions. The incorporation of leucine-3 H into the histone
and nonhistone proteins of the chromatins at 1 and 24 hr is
given in Tables 2 and 3. The results are expressed as ratios,
because the supply of amino acids to the hepatomas and their
utilization in these tissues are probably not comparable with
liver. The data show that after 1 hr both hepatomas show a
slightly greater incorporation
of labeled amino acid into
nuclear protein relative to the incorporation into total-cell
protein. The nuclear protein of liver, on the other hand, shows
a smaller uptake relative to total-cell incorporation. These
differences disappear, however, after 24 hr. These findings are
in agreement with earlier reports which showed that tumor
nuclei in general have a greater protein synthetic capacity than
tumor cytoplasm (7, 8). Because the observed changes are
small and we are comparing cell populations which may take
up and utilize amino acids differently and which may have
different contents of inactive structural protein and necrotic
tissue, the interpretation of this data must be treated with
caution. The incorporation of label into liver total-cell protein
drops over 40% in from 1 to 24 hr, but the incorporation of
label into both hepatomas shows little change with time,
1172
3.5
3.0
NORMAL
LIVER
2.5
2.0
S
z
1.0
0.5
10
15
FRACTION
20
25
NO.
3.5
3.0
HEPATOMA
5123C
2.5
2.O
o
S 1.5
O.OSM
O.31M
HaCI
NoCI
0.5
10
15
FRACTION
20
25
NO.
Chart 2. Fractionation of total chromosomal proteins of normal liver
and hepatoma 5123C into histone and nonhistone proteins on a column
of SE-Sephadex C-50 (0.9 x 25 cm). Buffer, 0.01 M sodium acetate (pH
5.0) in 5 M urea; fraction size, 4.5 ml; load, normal liver, 9.9 mg
protein and hepatoma 5123C, 7.2 mg protein. The 1st major peak is the
nonhistone proteins; the 2nd peak is the histones. (Histone Fl does not
elute with the histone peak but can be removed from the column by
washing with 0.7 M NaCl.) The elution profile of the proteins from
hepatoma 96ISA was the same as that from normal liver.
indicating a reduced amount of protein turnover. The
incorporation of label into liver nuclear proteins follows the
same pattern but drops only 30% in from 1 to 24 hr, while the
nuclear proteins from hepatoma 5123C show a turnover
approaching that of the liver proteins. The most striking result
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Study ofChromatin from Hepatomas
of the data is the increase in the incorporation of label into DISCUSSION
histones relative to nonhistone proteins of both hepatomas at
The findings of this study show that the template activity
both time periods. This result is in agreement with the findings
of many workers that histone synthesis is greatly increased and electrophoretic profile of the major nonhistone proteins
in tumor cells (7-9, 16, 41). Like total cell protein, the of the highly differentiated hepatoma 96ISA chromatin are
nonhistone proteins of liver turn over rapidly and lose about indistinguishable from those of normal liver. Because these 2
45% of their label in 24 hr, as was noted in earlier studies (11, properties of chromatin reportedly are altered in poorly
21). The nonhistone proteins of both hepatomas, however, differentiated tumors (13, 21, 22, 37) and are also shown to
show a reduced turnover. These general findings were also be altered in the analysis of the 5123C hepatoma chromatin
made in preliminary experiments, which used an amino reported here, our data suggest that changes in these properties
acid-14C mixture as label instead of leucine-3H.
in anaplastic tumors may correlate more with malignant
progression than with changes essential for neoplastic transfor
mation. The findings also show that the 9618A hepatoma
chromatin does have an altered content of nonhistone protein
and RNA, an altered rate of amino acid incorporation into
histone protein, and an altered turnover of the nonhistone
proteins, compared with normal liver chromatin. These 3
differences are reportedly shared by tumor chromatins in
general (8, 9, 11, 16, 37, 41, 46) and are likewise confirmed
by analysis of the 5123C hepatoma chromatin.
The biological significance of the increased nonhistone
protein and RNA content in the 2 hepatomas, as well as in
tumor chromatin in general, is not known. It has been
suggested that the nonhistone proteins play a specific role in
the control of transcription by opening up stretches of the
genome which are nonspecifically repressed by histone (14,
39, 43). This theory predicts that greater amounts of non
histone protein would probably increase the template activity
of the chromatin. Within the limitations of an in vitro assay
system, we do find an increase in RNA synthesis from the
5123C chromatin. On the other hand, the 96ISA chromatin,
which contained only a little less extra nonhistone protein, did
not show any significant increase in template activity. We are
unable to explain this discrepancy on the basis of our present
data. It is possible that a complex process of both repression
and derepression is occurring in the hepatomas. The theory
that normally expressed genes are repressed in tumor cells is
well established, since tumors in general lack many enzyme
systems found in normal tissues. The possibility that both
repression
and derepression occur simultaneously has recently
Fig. 2. Electrophoretic patterns of chromosomal nonhistone proteins
from normal liver and hepatomas on 7.5% acrylamide gels, pH 2.7. Gel been suggested by the work of Shearer and Smuckler (36).
Increased amounts of nonhistone protein may also con
on left shows proteins from normal liver, middle gel shows proteins
tribute to the greater insolubility of both hepatoma chromafrom hepatoma 96 ISA, and gel on right shows proteins from hepatoma
5123C. About 70 ng of protein were applied per gel.
tins. However, other considerations, such as a change in the
I
Table 2
Ratio of incorporation of leucine-3 H into whole-cell versus nuclear protein and into
nonhistone versus histone protein of liver and hepatomas
The ratios were calculated from the specific activities of the separate protein fractions
expressed as cpm/mg protein. The specific activity of whole-cell protein was determined on the
filtered homogenate of the tissue. Specific activities were determined on protein fractions from
the pooled tissue of 1 to 4 rats. Each ratio represents the average of 2 to 5 ratios determined
independently.
Tissue
Whole-cell :nuclear protein
Nonhistone: histone protein
1-hr label
1-hr label
0.03°
Liver
0.83 ±0.04
Hepatoma 9618A
Hepatoma 5 123C1.07±0.84 ±0.170.84
24-hr label
±0.06
0.87 ±0.09
1.41 ±0.08
1.06 + 0.213.47*0.11
1.77 ±0.322.52
24-hr label
±0.28
1.42 ±0.09
1.70±0.40
" Mean * S.E.
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1973
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E. A. Arnold, M. M. Buksas, and K. E. Young
Table 3
Ratio of incorporation ofleucine-3Hat ! hr versus 24 hrfor whole-eel!, nuclear,
nonhistone, and histone protein of liver and hepatomas
Ratios were calculated by dividing the specific activity of the protein fraction labeled for 1
hi by the specific activity of the fraction labeled for 24 hr. See the explanatory paragraph in
Table 2 for additional details.
TissueLiver
protein1.75
protein1.42
protein1.84
protein1.33
±0.23°
±0.06
±0.03
±0.21
1.30ft
1.32b
1.10±0.11
1.11 ±0.09
Hepatoma9618A
1.02 ±0.16Histone1.06 ±0.35
1.14±0.10Nuclear 1.33 ±0.29Nonhistone
Hepatoma5123CWhole-cell
" Mean ±S.E.
b Only 1 determination of the specific activity of hepatoma 9618A histone and nonhistone
proteins labeled for 24 hr could be used for calculating the ratio.
proportions of euchromatin and heterochromatin (12), molec
ular weight, or the presence of contaminating material, may
play important roles.
We were concerned that the additional nonhistone protein
we observed in tumor chromatin might be artifact, since
Grunicke et al. (15) reported that they could not find any
difference between the protein content of liver and 5123C
hepatoma chromatin. Several investigators (19, 38) have
suggested that many of the acidic proteins found in chromatin
preparations may be artifactual, having arisen from contamina
tion by cytoplasmic proteins during the isolation procedure. It
has also been suggested that the increased amounts of nuclear
residual proteins observed in many tumors may also be
artifactual (42). Experiments done in this laboratory have
shown, however, that the nonhistone
proteins of the
hepatoma, as well as liver prepared from nuclei which had
been mixed with a leucine-3H-labeled postnuclear (cyto
plasmic) supernatant were only slightly contaminated with
radioactive protein. Likewise, nonhistone proteins isolated
from chromatin that had been mixed with a labeled nuclear
sap preparation exhibited only a trace of radioactivity. In
addition, washing of the nuclei with Triton X-100, which
removes the outer nuclear membrane (a possible contaminant
in chromatin preparations), did not reduce the nonhistone
content. These types of test experiments have their limita
tions, however, and cannot assess the effect of all types of
processes in which contamination might occur. The wide
variability of the total protein values for the hepatoma 5123C
chromatin from one preparation to another and the slight
discoloration noted in samples of hepatoma chromatin suggest
that some contamination could nonetheless be occurring.
The incorporation data of the chromosomal proteins we
report are consistent with previous findings that histones are
synthesized at a much greater rate in tumors than in normal
tissues (7-9, 16, 41). Because there was variability in the
results from one tumor experiment to another, however, it is
impossible to determine whether there are significant differ
ences in the rate of incorporation of amino acid into the
chromosomal proteins between the 2 tumors themselves.
The significance of the increased incorporation of amino
acid into the histones of the hepatomas is not resolved by our
data. Because the mitotic rate of the hepatoma 9618A as
measured by growth rate and thymidine-3 H incorporation into
1174
DNA is very low (31), it is difficult to see how increased cell
division alone could account for the increase in incorporation.
The reduced turnover of nonhistone proteins in the
hepatomas may be caused by a defect in the metabolism of
these proteins. As the nonhistone proteins have been linked
with the fine control of gene transcription, such a defect may
be related to altered cellular control systems seen in these
hepatomas and tumors in general. Dastugue et al. (Il)
reported that all of the nonhistone proteins apparently turn
over at the same rate in liver but not in the hepatoma studied.
Our finding of an increased template activity for the 5123C
hepatoma chromatin confirms that of Smith and Mora (37)
but does not agree with the data of Ornata and Ichii (30), who
did not find any difference between the activity of the
fast-growing tumor and of liver chromatin. Because the
physical state of a chromatin preparation can affect its
template activity (17), it is possible that this discrepancy
results from the use of different methods of chromatin
preparation. The significance of these assays is also question
able because of recent findings concerning the validity of using
a bacterial RNA polymerase with mammalian chromatin (10,
20).
Although we could not detect qualitative differences
between the electrophoretic
pattern of the nonhistone
proteins of the highly differentiated tumor and that of liver,
minor qualitative differences could exist. Deletions or new
synthesis of very minor protein fractions, such as individual
repressor or activator proteins, if they exist in mammalian
cells, would probably not be seen on the gels. It is possible
that minor changes in proteins such as these could be the
fundamental changes necessary for malignant transformation.
The results do suggest that the increased amount of
nonhistone protein found in the 9618A chromatin does not
come primarily from the synthesis of new protein species but
must result from a quantitative increase in nonhistone protein
species that are present in liver. The assumption is made,
however, that the additional nonhistone protein is not
selectively lost in the isolation procedure.
ACKNOWLEDGMENTS
We wish to thank Dr. Harold P. Morris of Howard University for
supplying the original stock of hepatoma-bearing animals.
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Study of Chromatin from Hepatomas
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Rate. Cancer Res., 32: 1775-1784,1972.
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E. A. Arnold, M. M. Buksas, and K. E. Young
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CANCER
RESEARCH
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VOL.
33
A Comparative Study of Some Properties of Chromatin from
Two ''Minimal Deviation'' Hepatomas
Eugene A. Arnold, Mary Margaret Buksas and Keith E. Young
Cancer Res 1973;33:1169-1176.
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