Download Elevated expression and altered pattern of

Carcinogenesis vol.19 no.10 pp.1777–1781, 1998
Elevated expression and altered pattern of activity of DNA
methyltransferase in liver tumors of rats fed methyl-deficient diets
N.G.Lopatina, B.F.Vanyushin1, G.M.Cronin and
L.A.Poirier2
Division of Nutritional Toxicology, National Center for Toxicological
Research, NCTR Drive, Jefferson, AR 72079, USA
1Present
address: A.N.Belozersky Institute of Physico-Chemical Biology,
Moscow State University, Moscow 119899, Russia
2To
whom correspondence should be addressed
Email: [email protected]
DNA methyltransferase (MTase) activity in nuclear extracts
from neoplastic and preneoplastic livers of rats fed a
methyl-deficient diet (MDD) is elevated compared with that
seen in the livers of control rats. Nuclear proteins were
prepared in the presence of protease inhibitors including
trans-epoxy succinyl-L-leucylamido-(4-guanido)butane and
were fractionated by isoelectric focusing. In normal, control
liver, two distinct MTase fractions were observed. In MDDinduced malignant liver, a third fraction, in addition to the
previous two, was also seen. Both the DNA substrate and
the cytosine site specificities of the third MTase fraction
differ from those of the other two fractions. The distinct
MTase activity in liver tumor has significantly more de novo
MTase activity than do the MTase fractions of normal,
control liver. Thus, normal and neoplastic rat livers differ
in DNA MTase fractionation patterns and site specificities.
The altered DNA MTase activity observed in rat liver
tumors caused by MDDs may be one of the critical factors
contributing to cancer formation through abnormal DNA
methylation.
Introduction
Methyl-deficient diets (MDDs) cause cancer (1–3) and DNA
under-methylation in rodent liver (4–10). Since DNA methylation plays a significant role in the regulation of transcription,
DNA replication and DNA repair (11), it is postulated that
abnormal DNA methylation may play an essential role in
carcinogenesis (12–15). Currently, little is known about the
nature and specificity of DNA (cytosine-5)-methyltransferase
(EC 2.1.1.37) (MTase) in malignant tissues (14–17) and about
the extent to which the enzyme may be responsible for the
abnormal DNA methylation found in cancer cells. Furthermore,
the effects of stringent dietary methyl deficiency on hepatic
DNA MTase require further investigation. Previous studies
have shown that the chronic administration of methyl-deficient
diets to rats leads to increased MTase activity both in preneoplastic liver and in the resultant tumors (7,9,10). The present
study provides evidence showing that tumors produced by
MDD display elevated expression and activity of DNA
Abbreviations: E-64, trans-epoxy succinyl-L-leucylamido-(4-guanido)butane;
m5C, 5-methyldeoxycytidine; MDD, methyl-deficient diet; MSD, methylsufficient diet; MTase, DNA(cytosine-5)-methyltransferase; PMSF, phenylmethylsulfonyl fluoride; SAM, S-adenosylmethionine; TLCK, N-tosyl-L-lysine
chloromethyl ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone.
© Oxford University Press
MTase(s), as well as changes in the patterns and site specificity
of these enzymes. Thus, hepatocarcinogenesis by methyl deficiency is accompanied by the appearance of new DNA
MTase(s) with a modified site specificity.
Materials and methods
Animals and feeding regimens
Male weanling F344 rats (50–60 g) were divided into two groups, and fed
one of two amino-acid-defined diets developed by Mikol et al. (1). One group
received the methyl-sufficient diet (MSD), while the other was fed the MDD
(1). MSD contained 0.5% DL-methionine and 0.2% choline chloride. MDD
contained no methionine or choline, but was supplemented with an amount
of DL-homocystine equimolar to the methionine removed. Each diet contained
folic acid (5 mg/kg) and vitamin B12 (50 µg/kg). Normal liver was obtained
from control animals fed MSD for 6, 64 and 82 weeks. Liver tumors were
obtained from rats fed MDD for 82 weeks, while preneoplastic liver was
obtained from rats fed MDD for 6 and 64 weeks. The rats were killed with
CO2, the livers were excised and immediately frozen in liquid nitrogen, and
then kept at –70°C until used for the isolation of nuclei, RNA and DNA.
The patterns of MTase activities obtained from normal and tumorous liver
were also compared in a second experiment. In this case, the MDD consisted
of a semi-synthetic soy bean-based diet previously used in this laboratory (9).
This diet contained 0.18% methionine, and lacked both choline and folic acid;
the corresponding control diet contained 0.4% methionine, 0.3% choline and
2 mg/kg folic acid (9). Both the liver tumors and the control livers used in
the second study were obtained from animals fed the respective MDD and
MSD for 54 weeks. No sulfa drugs or carcinogens were used in any of these
investigations. In both studies, care was taken during autopsy to separate the
tumor tissue from the preneoplastic liver. However, the presence of some nonneoplastic tissue in the tumor samples cannot be completely excluded. The
effects of both types of MDD on growth inhibition and on liver pathology
have been amply described elsewhere (1,3,5,9,10). As shown in Results, the
effects of both deficient diets on tumor DNA MTase distribution were
virtually identical.
DNA isolation
Frozen rat liver tissue (50–60 mg) was homogenized, using a glass homogenizer
with a teflon pestle, in 600 µl of a solution (pH 8.3) containing 25 mM
EDTA, 100 mM NaCl, 1% SDS and 150 µg/ml of proteinase K (Boehringer
Mannheim, Indianapolis, IN). Homogenates were incubated overnight at 37°C,
treated with RNase A (10 µg/µl) (Boehringer Mannheim) for 1 h at 37°C,
and DNA was isolated using a Puregene DNA isolation kit (Gentra Systems,
Plymouth, MN). DNA was quantitated spectrophotometrically at 260 nm, and
its purity was determined by the ratio of absorbance at 280 to 260 nm.
Assay of methylation status of DNA MTase gene
Eight microgram DNA aliquots were hydrolysed with 10 U of HpaII restriction
endonuclease (Gibco BRL, Gaithersburg, MD). DNA hydrolysates were
fractionated by electrophoresis in 0.9% agarose (16) and transferred to Hybond
N1 (Amersham, Amersham, UK) by alkaline blotting as recommended by
the manufacturer. The membranes were prehybridized for 1 h as described
(16), and then incubated with a random primed [α-32P]dCTP-labeled probe
at 60°C for 18 h. The 4423 bp C-terminal murine cytosine DNA MTase gene
fragment cut from the pBlueScript construct containing a full copy of this
gene (a gift from Dr T.H.Bestor) by BamHI restriction endonuclease (Gibco
BRL) and isolated by electrophoresis in a 0.9% polyacrylamide gel was used
as a probe. The membranes were washed (16) and exposed to Kodak X-Omat
AR film for 14 days at –70°C. The progressive loss of high molecular weight
material with the accumulation of smaller molecular weight bands in DNA
samples hydrolyzed by HpaII enzyme is indicative of DNA demethylation at
Cm5CGG sites.
RNA isolation and northern blots
mRNA was isolated from 0.5 g frozen liver samples using a FastTrack mRNA
isolation kit (Invitrogen, San Diego, CA). RNA was measured by its absorbance
at 260 nm. Aliquots of 1 µg liver mRNA from control and experimental rats
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N.G.Lopatina et al.
were applied to Hybond as described by the manufacturer. Hybridization with
a random primed [α-32P]dCTP-labeled 4423 bp C-terminal murine DNA
MTase gene fragment was performed in 0.25 M Na2HPO4 (pH 7.2), 7% SDS
at 64°C for 18 h, and the membranes were washed twice for 15 min each in
0.02 M Na2HPO4, 5% SDS and twice for 15 min each in 0.02 M Na2HPO4
1% SDS at 64°C. The blots were exposed at –70°C to Kodak X-Omat film
for 1–2 days.
Isolation and determination of DNA MTase activity
Rat liver nuclei and nuclear extracts were isolated as described (17,18). The
protein concentrations both in nuclear extracts and in samples obtained by
isoelectric focusing were determined as previously described (19). The DNA
MTase reaction conditions were such that both the methyl donor and the DNA
template were in excess, so that the extent of methyl group incorporation was
dependent on the level of DNA MTase activity. Extracts were incubated for
4 h at 37°C in a 130 µl reaction mixture containing 10 mM Tris-HCl buffer
(pH 7.4), 5 mM EDTA, 10 µg of calf thymus DNA (Sigma, St Louis, MO),
and 1 µCi of S-adenosyl-L-[methyl-3H]methionine (SAM, sp. act. 7.9 Ci/mmol;
NEN, Boston, MA). The samples were applied to Whatman DE filters, and
DNA radioactivity was quantified by liquid scintillation counting; enzyme
activity was expressed as cpm/mg protein.
DNA MTase fractionation and the site specificity assay
To fractionate DNA MTases, the nuclear proteins were precipitated by the
addition of solid ammonium sulfate to the nuclear extracts obtained above
(600 mg/ml, or 80% saturation) (17). The precipitates were collected by
centrifugation at 105 000 g for 1 h and dialyzed as previously described (20).
The resulting protein preparations were processed on a Rotofor Cell column
(Bio-Rad, Hercules, CA) as described by the manufacturer. Twenty 4 ml
fractions of each protein preparation applied were obtained as the result of
the isoelectric focusing for 6 h in the presence of ampholytes at pH 3–10. All
procedures on DNA MTase isolation (tissue homogenation, nuclei isolation,
protein precipitation and dialysis, and electrophoresis) were carried out at 4°C
and in the presence of our standard set of protease inhibitors, i.e. 0.2 mM
phenylmethylsulfonyl fluoride (PMSF) and 1 µg/ml leupeptin (17,20).
In a second set of studies, MTase was prepared from tumors derived from
rats fed the semi-synthetic MDD (9); in this case the DNA MTase isolation
procedure was modified to include, in addition to the PMSF and leupeptin
used as above, the protease inhibitors trans-epoxy succinyl-L-leucylamido(4-guanido)butane (E-64), N-tosyl-L-lysine chloromethyl ketone (TLCK) and
N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), each at 10 µg/ml as
previously described (18). The studies with the additional protease inhibitors
were prompted by the findings of Xu et al. (18), showing that they effectively
inhibited proteolysis during the isolation and partial purification of DNA
MTase from tissue. All five protease inhibitors were purchased from Boehringer
Mannheim.
As described above, aliquots of each fraction obtained by isoelectric
focusing were used for the determination of DNA MTase activity. To
distinguish between the site specificities of the DNA MTases found, 10 µg
calf thymus DNA was incubated with 50 µl aliquots of each peak containing
fraction in the presence of [methyl-3H]SAM under the conditions already
described. The labeled DNA was hydrolyzed by treatment with 66% formic
acid 1 1% diphenylamine at 30°C for 18 h. The DNA pyrimidine isopliths
liberated were fractionated by thin-layer DEAE-cellulose chromatography (21),
and the radioactivity in monopyrimidine (pCp1pm5Cp1pTp), dipyrimidine
[(C2p3 1 pm5CpCp 1 pCpm5Cp 1 m5C2p3) 1 (pCpTp 1 pTpCp 1
pm5CpTp 1 pTpm5Cp) 1T2p3] and tripyrimidine (Py3p4) isopliths was
measured. This method allowed us to determine the radioactivity not only of
all newly formed 5-methyldeoxycytidine (m5C) residues in DNA pyrimidine
isopliths but also of possible thymine residues resulting from the enzymatic
deamination of m5C formed during in vitro DNA methylation (22).
Results
DNA MTase activity
The relative DNA MTase activities in nuclear extracts from
the preneoplastic livers of rats fed MDD for 6 weeks and from
the tumorous livers of rats fed MDD for 86 weeks were 130 6
8.6 and 184 6 8.7% (mean 6 SD), respectively, of that found
in the livers of control rats fed MSD (100 6 10.1). Thus,
carcinogenic MDD produces a significant increase in the total
nuclear DNA MTase activity. This observation is consistent
with previous results on the general increased DNA MTase
activity in neoplastic and preneoplastic tissues (7,12,13),
including the livers of methyl-deficient animals (7,9,10).
1778
Fig. 1. Slot blots of rat liver mRNA hybridized with the 32P-labelled
4423 bp murine DNA MTase gene probe. From top to bottom, the lanes
contain: mRNA from control rats fed MSD for 6 weeks; mRNA from rats
fed MDD for 6 weeks; mRNA from rats fed MDD for 64 weeks; and
mRNA from tumors of rats fed MDD for 82 weeks. The DNA MTase to
β-actin signal (mRNA) ratio in neoplastic livers was 2.1–2.9-fold higher
compared with that in normal livers.
Fig. 2. Southern blot of rat liver DNA hydrolysed by HpaII restriction
endonuclease and hybridized with the 32P-labeled 4423 bp murine DNA
MTase gene probe. Lane 1, DNA from control rats fed MSD diet; lanes 2
and 3, DNA from rats fed MDD diet for 6 weeks; lane 4, DNA from liver
tumors of rats fed MDD for 82 weeks.
DNA MTase gene expression and methylation
A significant increase in liver DNA MTase expression was
found in rats fed MDD for 6 weeks (Figure 1). A lesser
increase was observed after 64 weeks of MDD feeding. The
difference in MTase expression between 6 and 64 weeks’
feeding of MDD cannot now be explained; however, it is
consistent with other biochemical alterations whose magnitude
is greater in the early stages of MDD carcinogenesis than in
the later stages (2,4). Finally, liver tumors caused by MDD
were characterized by high DNA MTase expression (Figure
1). The DNA MTase gene sequence in preneoplastic (Figure
2, lanes 2 and 3) and tumorous (Figure 2, lane 4) rat livers is
less resistant to HpaII restriction endonuclease than is the
same sequence in normal liver (Figure 2, lane 1). Thus, the
internal C residues in CCGG sequences in the rat DNA MTase
gene are under-methylated in preneoplastic and neoplastic
livers compared with the corresponding internal C residues in
normal liver. This coincides with the under-methylation of
other genes in the liver carcinomas of rats fed MDD (6–9).
Thus, the hypomethylation of the DNA MTase gene in the
livers of MDD rats correlates well with an elevated transcription
of this gene.
Multiplicity of DNA MTase activities
Fractionation by isoelectric focusing of the hepatic nuclear
proteins obtained from control rats showed two protein frac-
Expression and pattern of activity of DNA transferase
Fig. 3. The distribution patterns of DNA MTase activities toward calf thymus and Drosophila DNA following isoelectric focusing of rat liver nuclear
proteins. (A) Normal liver without E-64. (B) Liver tumor without E-64. (C) Normal liver with E-64. (D) Liver tumor with E-64. cpm, radioactivity
incorporated from (methyl-H3)SAM into DNA in presence of MTase protein fraction; pI, protein isoelectric point. The studies with E-64 protease inhibitor
were prompted by the findings of Xu et al. (18) that this compound was an effective inhibitor of proteolysis during the isolation and purification of DNA
MTase from mouse tissue.
tions, with pI values at ~4.3 and 7.3, respectively, possessing
significant MTase activity (Figure 3A and C). The same two
fractions were obtained regardless of whether the DNA MTase
was isolated using the standard mixture of protease inhibitors
(20) or the more complete set of such inhibitors including
E-64 (18) (Figure 3A and C). As is commonly the case with
mammalian DNA MTase, the two DNA MTase fractions
obtained from control livers exhibited greater enzymatic
activity toward calf thymus DNA, which is methylated, than
toward Drosophila DNA, which is not (Figure 3). In addition,
the high proportion of monopyrimidine isopliths (66%)
obtained from the hydrolyzed calf thymus DNA methylated
in vitro with the MTase fraction at pI 4.3 indicates methylation
of cytosine residues located between two purines in the DNA
sequence (Table I). In contrast, the distribution pattern of
radioactivity incorporated from 3H-methyl groups from SAM
into DNA pyrimidine isopliths obtained from calf thymus
DNA treated with MTase at pI 7.3 that had been isolated from
control livers was quite different (Table I); in this case
the proportion of radioactivity incorporated into the DNA
tripyrimidine isopliths was 10 times greater than that seen
with the MTase isolated at pI 4.3 (Table I). Thus, the pI 7.3
Table I. Distribution of radioactivity among pyrimidine isopliths (Pyn pn11)
in calf thymus DNA methylated in vitro in the presence of S-adenosyl-L[methyl-3H]methionine by DNA MTase fractions isolated from the rat liver
nuclear extracts by isoelectric focusing
Protein fraction pI
Radioactivity (%) incorporated into DNA pyrimidine
isopliths
Mono
Di
Tri
31.9
29.9
2.1
22.1
Normal liver from rats fed MSD
4.3
7.3
66.0
48.0
Neoplastic liver from rats fed MDD for 82 weeks
4.3
62.2
32.4
7.3
50.8
30.0
8.5
73.3
15.7
5.4
19.2
11.0
SD values were not higher than 5% of data represented; n 5 8–10.
fraction may represent a peptide that is different from that
seen at pI 4.3.
In contrast to normal liver, the nuclear extracts obtained
1779
N.G.Lopatina et al.
from hepatic tumors in methyl-deficient rats showed three
different fractions with MTase activity, viz. at pI 4.3, 7.3 and
8.5, respectively, following fractionation by isoelectric focusing
(Figure 3B and D). Again, identical results were obtained
regardless of the combination of protease inhibitors used
(Figure 3B and D) (17,18). The tumor enzyme fractions at
pI 4.3 and 7.3 were equivalent to the corresponding fractions
obtained from the control livers. They each methylated calf
thymus DNA better than Drosophila DNA (Figure 3). The
tumor pI fractions at 4.3 and 7.3 each produced distribution
patterns of radioactivity among the three pyrimidine isopliths,
obtained by hydrolysis of the substrate DNA, closely
resembling the patterns produced by the corresponding MTase
pI fractions from control livers (Table I). However, the DNA
MTase fraction at pI 8.5 found in neoplastic liver had no
equivalent in normal liver (Figure 3). Unlike the fractions at
pI 4.3 and 7.3, the tumor MTase at pI 8.5 had greater enzymatic
activity toward Drosophila DNA than toward calf thymus
DNA (Figure 3). In its radioactive distribution patterns among
the pyrimidine isopliths, the tumor DNA MTase at pI 8.5 also
exhibited significant differences in site specificity compared
with the fractions at pI 4.3 and 7.3 (Table I). Incubation of
calf thymus DNA with the pI 8.5 MTase fraction produced
greater incorporation of 3H-methyl groups from SAM into the
monopyrimidine and less into the dipyrimidine isopliths than
did the corresponding MTase fractions at pI 4.3 and 7.3 (Table
I). Finally, the fact that the DNA MTase fraction at pI 7.3
preferentially methylates the C residues in the longer pyrimidine sequences indicates that it may preferentially methylate
CpNpG sequences in the DNA of animal cells (23).
We are greatly indebted to Dr T.H.Bestor for supplying us with the murine
DNA MTase clone and to Dr S.J.James for the tumors from methyl/folatedeficient rats. This work was supported by ORISE Fellowships to N.G.L.
and B.F.V.
Discussion
References
The major finding of this study is the detection by isoelectric
focusing of a new form of MTase activity in the liver tumors
of rats fed MDDs. This new MTase fraction is not found in
normal liver, exhibits altered substrate specificity, and, unlike
normal mammalian MTase, preferentially exerts de novo
methylase activity. Apparently new forms of MTase activity
have been previously observed in the lymphocytes of leukemic
cows (14), in HeLa cells (15) and in human thyroid goiters
(20). Unfortunately, the exact origin of the new activity of
MTase observed in liver tumor is unknown. It has also been
suggested that DNA MTases in L cells may exist in more than
one form that differ in site specificity and in sensitivity to
SAM analogs (24). Despite strong precautions, including
multiple protease inhibitors in the isolation medium, the
possibility of partial proteolysis still cannot be completely
excluded. Thus, some DNA MTase fractions may appear
indirectly as a result of proteolysis of their common precursor
during isolation. Previous studies have shown that the proteolytic removal of a large N-terminal sequence from murine
DNA MTase does not change its maintenance methylation
activity but does appear to increase its de novo methylation
ability (25). If such proteolysis proceeds in vivo with the
formation of new DNA MTase activities, it could be a
mechansim of natural regulation of the enzyme site specificity
and de novo DNA methylation. On the other hand, it is very
likely that some nuclear DNA MTases observed here are
encoded by different genes. This seems to be the case for
some animal (13) and plant nuclear DNA MTases (26). Further,
mammals appear to have a gene for adenine DNA MTase (27),
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1780
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The present results are consistent with what is currently
known about the possible role of DNA methylation in carcinogensis. The high level of DNA MTase in tumor tissue is an
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the high tumor MTase activity noted in the present study was
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under-methylation of the DNA MTase gene. To the best of
our knowledge there is but one other publication describing
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Possibly the lower tumor incidence in transgenic animals with
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lessened amounts of abnormal enzymes. The development of
an altered form of DNA MTase with altered substrate specificity
during carcinogenesis would provide a plausible mechanism
linking the mutations (32–34), hypomethylation (6–13) and
hypermethylation (35,36) seen in DNA during cancer
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Received on December 1, 1997; revised on May 29, 1998; accepted on
June 4, 1998
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