Download Effect of Butyric Acid on Lung-colonizing Ability

(CANCER RESEARCH
46, 1244-1249,
March 1986]
Effect of Butyric Acid on Lung-colonizing Ability of Cloned Low-Metastatic
Lewis Lung Carcinoma Cells
Keizo Takenaga
Department oÃ-Chemotherapy, Chiba Cancer Center Research Institute, Nitona-cho 666-2, Chiba-shi, Chiba 280, Japan
ABSTRACT
The lung-colonizing ability of low-metastatic Lewis lung carci
noma cells (P-29) was enhanced by their in vitro treatment with
butyric acid and its sodium salt, sodium butyrate. Of the short
chain fatty acids tested, butyric acid was the most effective in
enhancing the lung-colonizing ability of P-29 cells; propionic acid
and valeric acid were slightly effective, but acetic acid and caproic
acid were ineffective. The enhancing effect of butyric acid on the
lung-colonizing ability of P-29 cells was reversible, indicating that
the result was the consequence of epigenetic alterations. Treat
ment of P-29 cells with butyric acid resulted in enhancement of
secretion of plasminogen activator, cellular cathepsin B activity,
and cellular adhesiveness. The phenotypes of cells treated with
butyric acid were compared with those of cells treated with
dimethyl sulfoxide, which was reported to enhance the lungcolonizing ability of P-29 cells. Significant differences were found
in the phenotypes, especially that of cellular adhesiveness; that
is, butyric acid enhanced mainly homotypic aggregation of the
cells, while dimethyl sulfoxide enhanced mainly heterotypic adhe
sion, such as adhesion to monolayers of endothelial cells. In
addition, butyric acid reversibly caused hyperacetylation of core
histones in P-29 cells, while dimethyl sulfoxide did not.
INTRODUCTION
The process of metastasis is complicated (1, 2), and so a
variety of properties of tumor cells are necessary to complete
the entire metastatic process. In recent years, by comparing the
properties of high-metastatic tumor cells with those of essentially
non- or low-metastatic cells, several properties have been found
to be involved in metastasis (1-3), including ability to adhere
heterotypically (1, 2, 4-6) and aggregate homotypically (1, 2, 79) and degradative enzyme activities, such as those of plasmin
ogen activator (3,10) and cathepsin B (3,11).
DMSO1 and other polar compounds are known to enhance
the lung-colonizing ability of cloned low-metastatic Lewis lung
carcinoma P-29 cells (12,13). This enhancement is accompanied
by increases in adhesiveness, secretion of plasminogen activa
tor, and activities of lysosomal enzymes including cathepsin B.
Although the mechanisms by which these polar compounds,
obviously nonphysiological reagents, exert their effects on P-29
cells are still unknown, investigations on this experimental sys
tem seem useful for determining not only the properties that are
correlated with colonizing ability, but also the mechanisms by
Received 7/30/85; revised 10/18/85; accepted 11/11/85.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1The abbreviations used are: DMSO, dimethyl sulfoxide; PBS, phosphatebuffered saline [138 mM sodium chloride:27 mM potassium chloride:8 mM dibasic
sodium phosphate;1.5 mw monobasic potassium phosphate (pH 7.4)]; HBSS,
Hanks' balanced salt solution; PMSF, phenylmethylsulfonyl fluoride.
CANCER
RESEARCH
which tumor cells acquire a high-colonizing potential.
DMSO and other polar compounds have been shown to cause
alterations in mammalian cells that are relevant to reverse trans
formation and induce differentiation of a variety of cell lines (1419). Butyric acid and its neutralized salt, sodium butyrate, have
also been shown to cause similar changes in mammalian cells
(14, 20-24). With these observations and the fact that butyric
acid is a naturally occurring fatty acid in mind, I examined the
effect of butyric acid on the lung-colonizing ability of P-29 cells.
The present study showed that butyric acid enhanced the
lung-colonizing ability of P-29 cells, that its effective concentra
tion was less than two-hundredths of that of DMSO, and that its
action was apparently different from that of DMSO.
MATERIALS AND METHODS
Reagents. 5-[125l]iodo-2'-deoxyuridine (5 Ci/mg) was purchased from
the Radiochemical Centre, Amersham, England. Benzoylcarbamylphenylalanylarginine-4-methyl-7-coumarylamide
was obtained from the
Peptide Research Foundation, Osaka, Japan. Human urokinase was
purchased from the Green Cross Corp., Osaka, Japan. Propionic acid,
butyric acid, valeric acid, and caproic acid were supplied by Nakarai
Chemicals, Kyoto, Japan, and acetic acid, sodium butyrate, and DMSO
were by Wako Pure Chemicals, Ltd., Osaka, Japan. Endothelial cell
growth supplement was purchased from Collaborative Research, Inc.,
Lexington, MA. Other chemicals were of the highest purity available.
Mice. Inbred male C57BL/6 mice 6 to 8 wk old were obtained from
Shizuoka Laboratory Animal Center, Hamamatsu, Japan.
Cell Line and Cell Culture. Cloned low-metastatic Lewis lung carci
noma P-29 cells (12,13, 25) were used in this study. They were cultured
in Dulbecco's modified Eagle's medium containing 10% heat-inactivated
(56°C,30 min) fetal calf serum, penicillin (100 units/ml), and streptomycin
(100 Mg/ml). Bovine pulmonary arterial endothelial cells, which were
obtained from the American Type Culture Collection, Rockville, MD, were
cultured in Dulbecco's modified Eagle's medium containing 10% heatinactivated fetal calf serum, endothelial cell growth supplement (5 /¿g/
ml), and insulin (25 ^g/ml). The cells were maintained in monolayer
culture and subcultured weekly. The cell lines were cultured at 37°Cin
a humidified atmosphere of 5% CO2 in air.
Assay of Lung-colonizing Ability. P-29 cells were detached from
culture dishes by 10-min treatment with 2 mw EDTA in PBS at 37°C.
Single-cell suspensions of the cells (1 x 10s cells per 0.2 ml of HBSS
per mouse) with greater than 95% viability, as assessed
staining, were injected into the tail vein of age-matched
mice. All mice were killed 16 days later, and their lungs
rinsed in water, and fixed overnight in Bouin's solution.
by trypan blue
male C57BL/6
were removed,
The number of
lung nodules was determined by counting parietal nodules under a
dissecting microscope.
Enzyme Assays. P-29 cells were cultured in the presence or absence
of butyric acid or DMSO for 5 days. Aliquots of the cells were scraped
off with a rubber policeman, washed by centrifugation, rapdily frozen at
-20°C, thawed, sonicated in a small amount of PBS, and used as an
enzyme source. Aliquots of the cells were washed with serum-free
medium, resuspended in serum-free medium, and cultured for a further
24 h for determination
VOL. 46 MARCH
of the secretion of plasminogen activator. Total
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EFFECT OF BITTYRATE ON COLONIZING
activity of cellular cathepsins B and L was determined by fluorometric
assay using benzyloxycarbamylphenylalanylarginine-4-m.ethyl-7-courn.ar-
to electrophoresis for 30 h at 400 V with cooling. Gels were stained with
0.2% Amido Black 10B in 10% acetic acid:45% methanol and destained
in 5% acetic acid:25% methanol.
ylamide as a substrate (26). One unit of activity is defined as the quantity
releasing 1 nmol of 7-amino-4-methylcoumarine
per min. Protein was
determined by the method of Lowry ef al. (27) with crystalline bovine
serum albumin as a standard. Plasminogen activator activity was deter
mined by the method of Saksela (28) with human urokinase as a
standard.
Assay of Adhesion to Monolayers of Endothelial Cells. P-29 cells
were cultured for 5 days in the presence or absence of butyric acid or
DMSO. In the last 20 h, the cells were radiolabeled by the addition of
0.5 fiC\ of 5-[125l]iodo-2'-deoxyuridine per ml of medium. The cells were
detached from culture dishes by 10-min treatment with 2 rtiM EDTA,
washed 3 times with PBS, and resuspended in complete medium. They
were then introduced onto completely confluent monolayers of endothelial cells and allowed to adhere without agitation at 37°C. After 5 min,
RESULTS
Effect of Butyric Acid on Lung-colonizing Ability of P-29
Cells. For examination of whether butyric acid enhances the
lung-colonizing ability of P-29 cells, the cells were treated with 1
mw butyric acid, for 1, 3, 5, and 7 days or with various concen
trations of butyric acid for 5 days, and then they were injected
i.v. into C57BL/6 mice. Sixteen days later, the mice were sacri
ficed, and the number of lung metastatic nodules was counted.
The results in Fig. 1 show that untreated P-29 cells formed a
few lung nodules, while butyric acid-treated cells formed many,
the unattached cells were carefully removed by three washings with
warm PBS, and the remaining adherent cells were lysed with 1 ml of 0.1
N NaOH. The lysate was collected, and its radioactivity was measured
(12,13).
Detachment Assay. P-29 cells were cultured for 5 days in the pres
ence or absence of butyric acid or DMSO. Then the culture dishes were
washed with serum-free medium and treated with 0.01% trypsin (Difco;
the number depending on the duration of treatment (Fig. 1a) and
the concentration of butyric acid (Fig. 1b). Thus, butyric acid
enhanced the lung-colonizing ability of P-29 cells. I also found
that sodium butyrate increased the lung-colonizing ability of P29 cells (data not shown). Butyric acid reduced the in vitro growth
of P-29 cells but was not cytotoxic; cell viabilities after exposure
to butyric acid were > 95%, as assessed by trypan blue staining.
Effect of Short Chain Fatty Acids on Lung-colonizing Ability
of P-29 Cells. In addition to butyric acid, I examined the effects
of short chain fatty acids such as acetic, propionic, valeric, and
caproic acid on the lung-colonizing ability of P-29 cells. These
fatty acids were tested at concentrations of 1 mw. As shown in
Fig. 2, butyric acid was the most effective, propionic acid and
valeric acid were slightly but significantly effective, and acetic
acid and caproic acid were ineffective.
Reversibility of the Effect of Butyric Acid on the Lungcolonizing Ability of P-29 Cells. For examination of whether the
effect of butyric acid on the lung-colonizing ability of P-29
cells was reversible, P-29 cells were cultured in medium contain
1:250). The dishes were placed on an orbital shaker rotating at 60 rpm.
After 10-min incubation, the cells released into the supernatant fluid were
collected and counted in a Model ZB Coulter Counter. All the remaining
attached cells were detached by vigorous pipeting and counted. The
number of cells released was calculated as a percentage of the total cell
number per culture dish.
Assay of Homotypic Aggregation. P-29 cells were cultured for 5
days in the presence or absence of butyric acid or DMSO. The cells
were detached from culture dishes by 10-min treatment with 2 mM
EDTA, washed 3 times with PBS, and resuspended in serum-free me
dium. Single-cell suspensions of P-29 cells (1 x 106 cells/ml) were gyrated
at 100 rpm at 37°C. After 15-min incubation, 1 ml of 2% glutalaldehyde
in PBS was added for fixation. The number of single cells was determined
in a hemocytometer.
Isolation of Histones. Histories were isolated by a modification of the
methods of Kastraba et al. (29) and Multhaup ef al. (30). Briefly, P-29
cells were detached by 10-min treatment with 2 mw EDTA and washed
with chilled PBS. All subsequent steps were carried out at 4°C. During
ing 1 rnw butyric acid for 5 days. Then some cells were cultured
in regular medium and others in medium containing 1 mw butyric
acid for 5 days further. On Days 5 and 10, the cells were injected
i.v. into syngeneic mice at a concentration of 1 x 105 cells/
mouse. The results in Table 1 show that untreated P-29 cells
formed a few lung nodules throughout the experiment, while
butyric acid-treated cells formed many; the numbers of lung
nodules per mouse were about 132 and 184 with cells treated
with butyric acid for 5 days and 10 days, respectively. On the
isolation of histones, 10 mM sodium butyrate was added to all solutions
to inhibit histone deacetylase (31). The cells were washed twice with 10
volumes of 0.14 M NaCI:0.01 M Tris-HCI (pH 7.0):0.1 mw PMSF:10 mw
mercaptoethanol and collected by centrifugation. The washed cells were
suspended in 10 volumes of distilled water containing 0.1 mM PMSF and
gently homogenized. The resulting crude nuclei were precipitated by
centrifugation at 1000 x g for 10 min. The crude nuclear pellet was
suspended in 5 volumes of 0.14 M NaCI:0.01 M Tris-HCI (pH 8.0): 1 mM
MgCI2:0.1 mM PMSF and mixed with an equal volume of 2% Triton X-
••
235) ••
—
2HEi
PMSF with centrifugation, and the final pellet was washed 3 times with
10 volumes of 0.05 M Tris-HCI (pH 8.0):0.1 mw PMSF. Histones were
extracted by adjusting samples to 0.4 N HjSCv, homogenizing the nuclei
by 30 strokes of a Potter homogenizer operated at 500 rpm, standing
the homogenate for 30 min on ice, and centrifuging it at 10,000 x g for
15 min. Histones were precipitated from the supernatant by adding 4
volumes of absolute ethanol and standing the mixture overnight at
-20°C. They were then collected by centrifugation at 10,000 x g for 30
min, lyophilized, dissolved in distilled water, and stored at -20°C.
154)••{il
(120(107-126)•'(52-92)/(O-
mM1'
5)
23),1
.
1Incubation
3
109)/(0-9)/
,
5
7
time (days)
,"»—•(4
0
0.1
0.25
0.5
Concentration
(mM)259)2
Fig. 1. Effect of butyric acid on lung-colonizing ability of P-29 cells. P-29 cells
were treated with 1 HIM butyric acid for various periods (a) and at various
concentrations of butyric acid for 5 days (b). Single-cellsuspensions of the cells (1
x 105per 0.2 ml HBSS per mouse)were injected i.v. Points, mean numbers of lung
nodulesin sevenmice;numbersin parentheses,rangesof numbersof lung nodules.
Acid:Urea:Triton
Pofyacrylamide
Gel Electrophoresis.
Histones
were analyzed on slabs (36 cm x 1 mm) of 12% polyacrylamide:5%
acetic acid:8 M urea:0.37% Triton X-100 gel (32). Histones (40 ng of
buffer were subjected
CANCER RESEARCH
(„1(173
239)(174-I92KX*•
(148-
100 in the same buffer. The nuclear suspension was stirred for 3 min
and then centrifuged at 1400 x g for 10 min. The nuclei were washed 3
times with 10 volumes of 0.05 M EDTA:0.05 M Tris-HCI (pH 8.0):0.1 mM
protein per lane) in 8 M urea:5% mercaptoethanol
ABILITY
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EFFECT OF BUTYRATE
200
"inn
(23-66)
(13-43)
(0-2)
(1
3
Acid
4
chain
6)
5
B
length
Fig. 2. Comparative effects of short chain fatty acids on lung-colonizing ability
of P-29 cells. P-29 cells were treated with 1 rriM concentrations of short chain fatty
acids for 5 days. The numbers 2, 3, 4, 5, and 6 indicate acetic, propionic, butyric,
valeric, and caproic acid, respectively. Single-cell suspensions (1 x 105 cells per
0.2 ml HBSS per mouse) were injected into mice i.v. Numbers in parentheses,
ranges of numbers of lung nodules in six mice. The broken line indicates the mean
number of lung nodules formed with untreated cells.
Table 1
Reversibility of the enhancing effect of butyric acid on the lung-colonizing ability
of P-29 cells
P-29 cells were cultured in medium with or without 1 rriM butyric acid. P-29 cells
treated with 1 mM butyric acid for 5 days were divided into two portions. One was
cultured in medium with 1 mM butyric acid, and the other in regular medium for a
further 5 days. On Days 5 and 10, the cells (1 x 105 per 0.2 ml HBSS per mouse)
were injected i.v. Values are mean numbers of lung nodules in seven mice.
Butyric acid (1 mM)
0-5 days
No. of lung nodules/mouse
5-10 days
10 days
5 days
0.7 (0-2)
184.7(161-221)
0.9 (0-2)
" Numbers in parentheses, range of numbers of lung nodules.
0 (Of
132.6(111-156)
Table 2
Effects of butyric acid and DMSO on various phenotypes of P-29 cells
P-29 cells were cultured in medium with or without 1 mM butyric acid or 280
mM DMSO for 5 days. Phenotypes were determined as described in "Materials and
Methods".
Phenotype
Lung-colonizing ability8
Untreated
0.5(0-2)
Butyric acid treated
180.7(168-191)
DMSO treated
134.8(58-184)
(range)
Total cathepsins B and L
0.11 ±0.016
0.21 ±0.01C
0.22 ±0.02o
activity (milliunits/mg
protein)
Plasminogen activator
(units/10s cells)
2.2 ±0.8
13.5 ±3.0d
8.1 ±0.8C
11.2 ±2.6
16.4 ±0.9"
31.6 ±4.7o
95.4 ±0.5
40.6 ±6.7e
Homotypic aggregation (% 81.9 ±1.1
21.7 ±1.3e
Attachment to endothelial
cell monolayers (% of
cells attached)
Detachment by trypsin (%
ABILITY
other hand, cells cultured in medium containing
5 days and then in regular medium for a further
only a few nodules. Thus, the enhancing effect
on the lung-colonizing ability of P-29 cells was
(150-191)
2
ON COLONIZING
8.7 ±1.5°
of cells detached)
65.3 ±O.l"
of single cells)
a Mean number of lung nodules after injection of 1 x 10s cells i.v.
6 Mean ±SE.
c Significantly different from the control at P < 0.001 as determined by Student's
t test.
" Significantly different from the control at P < 0.01.
" Significantly different from the control at P < 0.05.
CANCER
RESEARCH
butyric acid for
5 days formed
of butyric acid
reversible. The
effect of butyric acid on cell growth in vitro was also reversible
(data not shown).
Comparison of Properties of Butyric Acid-treated and
DMSO-treated P-29 Cells. I compared the properties of butyric
acid-treated P-29 cells with those of DMSO-treated cells. The
properties examined were the cell morphology, their activities of
degradative enzymes, their ability to adhere to monolayers of
endothelial cells, their resistance to trypsin-mediated detach
ment, and their ability to aggregate homotypically. As shown in
Table 2, 1 mM butyric acid was equally effective or slightly more
than 280 HIM DMSO in enhancing the lung-colonizing ability of
P-29 cells. Therefore, butyric acid is effective at less than twohundredths the effective concentration of DMSO. The morphol
ogies of untreated, DMSO-treated, and butyric acid-treated P29 cells are shown in Fig. 3 a, c, and e, respectively. Most
untreated cells were round, but some developed pseudopodia.
Upon treatment with DMSO, they became flattened and spindle
shaped. Upon treatment with butyric acid, they also became
flattened but appeared polygonal. Thus, butyric acid-treated P29 cells apparently differed in morphology from DMSO-treated
cells. The other properties are presented in Table 2. Treatment
of P-29 cells with butyric acid resulted in marked increases in
secretion of plasminogen activator and total activity of cellular
cathepsins B and L. The increases in cell-associated plasmino
gen activator activity and total activity of cathepsins B and L
were suppressed by simultaneous treatment with cycloheximide
(Table 3), suggesting that they were associated with de novo
synthesis of protein. Butyric acid was as effective as DMSO in
increasing these degradative enzyme activities. In addition to
these enzyme activities, slight increases in heterotypic adhesion
and in resistance to trypsin-mediated detachment were observed
on treatment of P-29 cells with butyric acid, although DMSO
was more effective than butyric acid in enhancing these pheno
types. On the other hand, butyric acid was more effective than
DMSO in enhancing the ability to aggregate homotypically. Fig.
3, b, d, and (, shows the abilities of untreated, DMSO-treated,
and butyric acid-treated P-29 cells to aggregate homotypically,
respectively. Conspicuously, DMSO-treated cells formed small
aggregates, whereas butyric acid-treated cells formed large
ones. Thus, butyric acid enhanced mainly homotypic aggregation
of P-29 cells, whereas DMSO enhanced mainly their heterotypic
adhesion. These observations show that the phenotypes of P29 cells enhanced by butyric acid and by DMSO are different.
Effects of Butyric Acid and DMSO on Histone Acetylation
Patterns. Total histones isolated from untreated, butyric acidtreated, and DMSO-treated P-29 cells were analyzed on
acid:urea:Triton gels (Fig. 4). In untreated cells, three forms of
histone H4 (representing non-, mono-, and diacetylated histone
H4, denoted as H40, H4,, and H42, respectively) and three forms
of histone H3 (H30, H3,, and H32) were observed (Lane 1). Upon
treatment with butyric acid, the amounts of histones H42, H43,
and H44 increased markedly with concomitant decreases in the
amounts of histones H40 and H4,. The increase in multiacetylated
histones was apparent as early as 8 h after the beginning of
butyric acid treatment (Lanes 2 to 5). Alteration in the subspecies
of histones H3 and H2B was also observed. However, upon
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EFFECT OF BUTYRATE
ON COLONIZING
ABILITY
Fig. 3. Morphological changes and the abil
ities of homotypic aggregation of P-29 cells
treated with DMSO and butyric acid, a and b.
untreated P-29cells; c and d, P-29 cells treated
with 280 HIMDMSO for 5 days; e and 1, P-29
cells treated with 1 mw butyric acid for 5 days.
a, c, and e, morphology, photographed at x 50.
b, d, and t, homotypic aggregation, photo
graphed at x 20.
in enhancement of their lung-colonizing ability. This change was
Table 3
Effect of cycloheximide on inductions of degradai/ve enzyme activities by
butyric acid
P-29 cells were cultured in medium with the indicated drugs for 5 days. Then
activities were determined.
TreatmentNone
Butyric acid (1 mu»)
Cycloheximide(100 ng/ml)
Butyric acid (1 HIM)+ cyclo
heximide (100 ng/ml)Total
0 Mean ±SE.
cathepsins
B and L activity
(milliunits/mg
protein)0.17
±0.02a
plasminogen activator
(units/mg
protein)2.6
±0.2
4.0 ±0.8
0 53 ±0.01
0.7 ±0.0
0.06 ±0.01
1.0 ±0.1
0.11 ±0.02Cell-associated
removal of butyric acid, the pattern of histone acetylation became
similar to that of untreated cells (Lane 6), indicating that the
acetylation of histones by butyric acid is reversible. On the other
hand, DMSO did not notably alter the acetylation pattern (Lane
7).
DISCUSSION
The present study demonstrated that treatment of P-29 cells
with butyric acid or its neutralized salt, sodium butyrate, resulted
CANCER
RESEARCH
accompanied by marked increases in degradative enzyme activ
ities and the ability of the cells to aggregate homotypically. The
activities of degradative enzymes, such as plasminogen activator
and cathepsin B, have been shown to be important in metastasis
(10, 11), especially in degrading basement membrane compo
nents (33, 34). The ability of tumor cells to aggregate homotypi
cally, which may facilitate their arrest in lung capillaries as tumor
emboli, has also been reported to be positively correlated with
their lung-colonizing ability (35, 36). Therefore, it is likely that,
upon treatment with butyric acid, P-29 cells gain high colonizing
ability by acquiring these phenotypes.
An interesting finding in the present study was that the phe
notypes of P-29 cells treated with butyric acid are different from
those of cells treated with DMSO. The most remarkable differ
ence was in the change in adhesiveness; that is, butyric acid
enhanced homotypic aggregation, whereas DMSO enhanced
heterotypic adhesion. The morphology of butyric acid-treated P29 cells was also different from that of DMSO-treated cells.
Thus, P-29 cells can be induced by different stimuli to become
two different cell types, both with high lung-colonizing ability.
The exact mechanisms by which butyric acid and DMSO
enhance expressions of the genes responsible for plasminogen
activator, cathepsin B, and adhesiveness are still unknown. Both
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EFFECT OF BUTYRATE
ON COLONIZING
1234567
t
t
f t
titettt
H33.
H32
H3,H30-
H44-
directly in the nucleus, altering the conformation of chromatin
and consequently gene expression, as suggested previously
(17). However, there still remains the possibility that the primary
target of the actions of these drugs is the cell membrane.
The effect of butyric acid on the lung-colonizing ability of P-29
u OA
"
cells was reversible. Similarly, the effect of DMSO on P-29 cells
has been reported to be reversible (12). Thus, the enhanced
lung-colonizing ability of P-29 cells induced by these drugs may
result from epigenetic rather than genetic alterations. It is well
known that cellular phenotypic diversity generates in tumor
progression. The generation of such diversity has been explained
by genetic mutation. However, in some cases, the phenotypes
of tumor cells, including metastatic ability, are relatively unstable
and easily drift towards either more or less malignant ones (42).
Recently, epigenetic mechanisms have been proposed to ac
count for rapid cellular phenotypic diversification. One of the
most interesting epigenetic mechanisms is DMA methylation,
which Kerbel ef al. (43) demonstrated using 5-azacytidine. In
principle, besides impermanent modifications of DMA, a variety
of postgenetic processes may account for the rapid phenotypic
diversification. The present study suggests that such postgenetic
modifications as histone acetylation are involved in epigenetic
mechanisms producing phenotypic alterations that are unstable.
Further studies on the present experimental system may enable us to detect cell surface molecules responsible for homotypic and heterotypic adhesion, the control mechanisms of the
expression of genes for these molecules, and the initial cellular
changes associated with progression from a low- to a highmetastatic phenotype.
I H1
siili«*
•Alii*«
H43
H42
H3
U2 B
H4
H4,
H40
Fig. 4. Acid:urea:Triton polyacrylamide gel analysis of historie subspecies of P29 cells Misiones were isolated as described in "Materials and Methods" from
untreated P-29 cells (Lane 1); cells treated with 1 HIM butyric acid for 8 h (Lane 2),
24 h (Lane 3), 72 h (Lane 4), and 120 h (Lane 5); cells treated with 1 mw butyric
acid for 120 h and then cultured in regular medium for a further 120 h (Lane 6),
and cells treated with 280 mm DMSO for 120 h (Lane 7). Histones were subjected
to electrophoresis for 30 h at 400 V on a 16- x 36- x 0.1 -cm slab gel.
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Therefore, both may affect a common pathway, either at a
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physiology in many normal and transformed mammalian cells
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expression in P-29 cells. On the other hand, DMSO may act
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Effect of Butyric Acid on Lung-colonizing Ability of Cloned
Low-Metastatic Lewis Lung Carcinoma Cells
Keizo Takenaga
Cancer Res 1986;46:1244-1249.
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